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Carbon nanotubes are shaped like fibres and can stimulate inflammation at the surface of the peritoneum when injected into the abdominal cavity of mice, raising concerns that inhaled nanotubes may cause pleural fibrosis and/or mesothelioma. Here, we show that multiwalled carbon nanotubes reach the subpleura in mice after a single inhalation exposure of 30 mg m(-3) for 6 h. Nanotubes were embedded in the subpleural wall and within subpleural macrophages. Mononuclear cell aggregates on the pleural surface increased in number and size after 1 day and nanotube-containing macrophages were observed within these foci. Subpleural fibrosis unique to this form of nanotubes increased after 2 and 6 weeks following inhalation. None of these effects was seen in mice that inhaled carbon black nanoparticles or a lower dose of nanotubes (1 mg m(-3)). This work suggests that minimizing inhalation of nanotubes during handling is prudent until further long-term assessments are conducted.

The use of alternating frequencies during stimulation can increase stimulation response of a medium voltage electrical stimulation unit (MVS) by increasing the rate of pH decline. Various combinations of frequency modulation were tested in experiment 1 to determine the treatment resulting in the greatest stimulation response; the lowest initial pH, fastest rate of pH decline, highest temperature at pH 6 and the highest number of carcasses with a pH of 6 by 25(o)C and the treatment achieving the highest number of carcasses in the pH temp window (temperature at pH 6 between 18-25(o)C). The objective meat quality of these treatments compared to an unstimulated treatment was tested in experiment 2. Modulating the frequency (Hz) across the 6 segmented electrodes of the MVS by 10, 15, 25, 10, 15, 25Hz (Treatment 6, using a pulse width: 2.5ms, current: 1A) resulted in the greatest stimulation response. This treatment may be suitable for abattoirs that hot bone sheepmeat and require fast pH declines to ensure minimal cold shortening of meat. However, this treatment did not result in the tenderer meat despite the higher stimulation response. This treatment may have induced a greater number of contractions overall and therefore a greater pH decline response but resulted in less myofibrillar disruption compared to the other treatments due to a concomitant decreased force of contraction thus reducing potential tenderisation. Maintaining a constant frequency of 15Hz (Treatment 1; pulse width: 2.5ms, current: 1A) resulted in a higher number of carcasses in the pH temp window required (temperature at pH 6 between 18-25(o)C) in part A (P<0.05) and in addition to the higher tenderness levels this treatment may be more appropriate to satisfy the overall demands of abattoirs using these systems. This paper has also demonstrated electrical stimulation results in tenderer meat compared to unstimulated meat even after 30d of ageing (2.53±0.4 compared to 2.85±0.1 for the loin (M. longissimus thoracis et lumborum) (P<0.01) possibly due to a protective benefit of stimulation on meat tenderness. Overall, no detrimental effects of modulating frequency were observed on drip loss or retail colour display despite a greater rate of colour change observed with the modulated frequency treatment and the longer aged product.

Functional evidence suggests that nitric oxide (NO) signalling in the rostral ventrolateral medulla (RVLM) is cGMP-dependent and that this pathway is impaired in hypertension. We examined cGMP expression as a marker of active NO signalling in the C1 region of the RVLM, comparing adult (>18 weeks) Wistar-Kyoto (WKY, n = 4) and spontaneously hypertensive rats (SHR, n = 4). Double label immunohistochemistry for cGMP-immunoreactivity (IR) and C1 neurons [as identified by phenylethanolamine N-methyltransferase (PNMT-IR) or tyrosine hydroxylase TH-IR)], or neuronal NO synthase (nNOS) neurones, failed to reveal cGMP-IR neurons in the RVLM of either strain, despite consistent detection of cGMP-IR in the nucleus ambiguus (NA). This was unchanged in the presence of isobutylmethylxanthine (IBMX; 0.5 mM, WKY, n = 4, SHR n = 2) and in young animals (WKY, 10-weeks, n = 3). Incubation of RVLM-slices (WKY, 10-weeks, n = 9) in DETA-NO (100 mum; 10 min) or NMDA (10 muM; 2 min) did not uncover cGMP-IR. In all studies, cGMP was prominent within the vasculature. Soluble guanylate cyclase (sGC)-IR was found throughout neurones of the RVLM, but did not co-localise with PNMT, TH or nNOS-IR neurons (WKY, 10-weeks, n = 6). Results indicate that within the RVLM, cGMP is not detectable using immunohistochemistry in the basal state and cannot be elicited by phosphodiesterase inhibition, NMDA receptor stimulation or NO donor application.

The supraoptic (SON) and the paraventricular (PVN) hypothalamic nuclei constitute major neuronal substrates underlying nitric oxide (NO) effects on autonomic and neuroendocrine control. Within these nuclei, constitutively produced NO restrains the firing activity of magnocellular neurosecretory and preautonomic neurons, actions thought to be mediated by a cGMP-dependent enhancement of GABAergic inhibitory transmission. In the present study, we expanded on this knowledge by performing a detailed anatomical characterization of constitutive NO-receptive, cGMP-producing neurons within the PVN. To this end, we combined tract-tracing techniques and immunohistochemistry to visualize cGMP immunoreactivity within functionally, neurochemically, and topographically discrete PVN neuronal populations in Wistar rats. Basal cGMP immunoreactivity was readily observed in the PVN, both in neuronal and vascular profiles. The incidence of cGMP immunoreactivity was significantly higher in magnocellular (69%) compared with preautonomic ( approximately 10%) neuronal populations (P < 0.01). No differences were observed between oxytocin (OT) and vasopressin (VP) magnocellular neurons. In preautonomic neurons, the incidence of cGMP was independent of their subnuclei distribution, innervated target (i.e., intermediolateral cell column, nucleus tractus solitarii, or rostral ventrolateral medulla) or their neurochemical phenotype (i.e., OT or VP). Finally, high levels of cGMP immunoreactivity were observed in GABAergic somata and terminals within the PVN of eGFP-GAD67 transgenic mice. Altogether, these data support a highly heterogeneous distribution of basal cGMP levels within the PVN and further support the notion that constitutive NO actions in the PVN involve intricate cell-cell interactions, as well as heterogeneous signaling modalities.

1. In diseases such as complex regional pain syndrome (CRPS), where neuropathic pain is the primary concern, traditional pain classifications and lesion descriptors are of limited value. To obtain better treatment outcomes for patients, the underlying pathophysiological mechanisms of neuropathic pain need to be elucidated and analysed so that therapeutic targets can be identified and specific treatments developed. 2. In the present review, we examine the current literature on sympathetically maintained pain (SMP), a subset of neuropathic pain, within the context of CRPS. Evidence from both human and animal studies is presented and discussed in terms of its support for the existence of SMP and the mechanistic information it provides. 3. We discuss three current hypotheses that propose both a site and method for sympathetic-sensory coupling: (i) direct coupling between sympathetic and sensory neurons in the dorsal root ganglion; (ii) chemical coupling between sympathetic and nociceptive neuron terminals in skin; and (iii) the development of a-adrenoceptor-mediated supersensitivity in nociceptive fibres in skin in association with the release of inflammatory mediators. 4. Finally, we propose a new hypothesis that integrates the mechanisms of chemical coupling and a-adrenoceptor-mediated supersensitivity. This hypothesis is based on previously unpublished data from our laboratory showing that a histological substrate suitable for sympathetic-sensory coupling exists in normal subjects. In the diseased state, the nociceptive fibres implicated in this substrate may be activated by both endogenous and exogenous noradrenaline. The mediating a-adrenoceptors may be expressed on the nociceptive fibres or on closely associated support cells.

Chromogranin A (CGA) is a major secretory protein present in the soluble matrix of chromaffin granules of neuroendocrine cells and tumours, such as phaeochromocytomas. CGA has several functions, some of which may be involved in the distinct phenotypic differences of phaeochromocytomas in patients with von Hippel-Lindau (VHL) syndrome compared to multiple endocrine neoplasia type 2 (MEN 2). In this study, we therefore compared tumour and plasma levels of CGA in patients with phaeochromocytoma associated with the two syndromes. We show that phaeochromocytomas from MEN 2 patients express substantially more CGA than tumours from VHL patients at both the mRNA (3-fold greater) and protein (20-fold) level. We further show that relative to increases in plasma catecholamines, patients with phaeochromocytomas associated with MEN 2 have higher plasma concentrations of CGA than those with tumours in VHL syndrome. These data supplement other observations that phaeochromocytomas in VHL compared to MEN 2 patients express lower amounts of catecholamines and other chromaffin granule cargo, such as chromogranin B and neuropeptide Y. Possibly the differences in tumour CGA expression may contribute to differences in secretory vesicle formation and secretion in the two types of tumours. Alternatively the differences in expression in CGA and other secretory constituents may reflect downregulation of the entire regulated secretory pathway in VHL compared to MEN 2 tumours.

PC12 cells are an established model for studying the role of N-methyl-d-aspartate (NMDA) receptors in excitotoxicity and function as multimeric assemblies of NR1 with at least one NR2(A-D) subunit. We examined NR1 splice variant and NR2 subunit expression in four PC12 cell-lines (ATCC, WEHI, Ordway and Flinders), correlated mRNA expression with protein expression, and used patch-clamp recordings to test functionality. PCR indicated strong expression of the NR1 splice variants NR1-2a and NR1-4a in all cell-lines, with the remainder weakly detected or absent. Real-time PCR showed variable levels of NR1 mRNA expression (all splice variants) between cell-lines and a significant increase in response to nerve growth factor in the WEHI and Ordway lines (NGF: 50ng/ml, 2.1- and 13.4-fold increases, respectively, P< or =0.05). mRNA for NR2A or NR2B was not detected in any PC12 cell-line. NR2C mRNA expression varied between lines and increased after NGF treatment (approximately 4-fold increase in WEHI and Ordway lines, P< or =0.05). In the Ordway line, NR2D mRNA was seen only after NGF treatment. Immunohistochemistry confirmed protein expression for NR1, NR2C and NR2D, and while fluorescence intensity changes in response to NGF paralleled mRNA responses, the degree of increase was of reduced magnitude. Whole-cell patch-clamping of NGF treated cells failed to detect functional NMDA receptors in any of the cell-lines. Our study demonstrates that in contrast to neurons from the CNS, PC12 cells do not express a normal complement of NMDA receptor-subunits, and this may be one factor limiting functional responses to NMDA/glutamate and consequently the use of PC12 cells as a neuronal model.

The intracellular second messenger, cyclic guanosine monophosphate (cGMP), a soluble guanylate cyclase (GC) product, is a primary mechanism for the transduction of a nitric oxide (NO)-initiated signal in the central nervous system. NO is produced from L-arginine by neuronal nitric oxide synthase (NOS), which is found in sympathetic preganglionic neurons of the intermediolateral cell column. This suggests the possibility that NO is a modulator of sympathetic nervous activity (SNA) through a cGMP-mediated mechanism. The aim of this study was to determine the effects of intrathecally injected membrane-permeant 8-bromo-cGMP and 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of the soluble form of GC, on arterial pressure in urethane anaesthetized (1.4 g kg(-1) I.P.) rats. The effects of intrathecal cGMP and ODQ on haemodynamic responses to haemorrhage were also investigated. Finally, L-arginine, the NO precursor, was also injected intrathecally, alone and in the presence of ODQ. Baseline mean arterial pressure (MAP) increased significantly after intrathecal 8-Br-cGMP injection (10 microl, 1, 3, 10, 30, 100 microm). A dose-effect relationship (1 microm to 100 microm) was also established (EC(50)=6.03 microm). During continuous haemorrhage, MAP was maintained in animals injected with 8-Br-cGMP, relative to the control group. Although no change in baseline MAP was observed as a result of intrathecal ODQ injection (10 microl, 100 mM), a greater rate of fall in MAP was observed during haemorrhage. Injecting L-arginine (10, 100, 1000 microm, 10 microl) showed a pressor effect that was consistent with the effect of the downstream messenger, cGMP. Furthermore, its pressor effect was blocked by ODQ pre-administration. The results indicate that cGMP increases blood pressure, and thus suggest that cGMP increases SNA. This supports the hypothesis that the sympathoexcitatory effects of spinal delivery of NO are mediated by a cGMP-dependent mechanism.

Phaeochromocytomas are rare neuroendocrine tumours that produce catecholamines and numerous secretory proteins and peptides, including neuropeptide Y (NPY), a vasoactive peptide with influences on blood pressure. The production of catecholamines and NPY by phaeochromocytomas is highly variable. This study examined influences of hereditary factors and differences in catecholamine production on tumour expression of NPY, as assessed by quantitative PCR, enzyme immunoassay and immunohistochemistry. Phaeochromocytomas included hereditary adrenaline-producing tumours (adrenergic phenotype) in multiple endocrine neoplasia type 2 (MEN 2), predominantly noradrenaline-producing tumours (noradrenergic phenotype) in von Hippel-Lindau (VHL) syndrome, and other adrenergic and noradrenergic tumours where there was no clear hereditary syndrome. NPY levels in phaeochromocytomas from VHL patients were lower (P<0.0001) than in those from MEN 2 patients for both mRNA (84-fold difference) and the peptide (99-fold difference). These findings were supported by immunohistochemistry. NPY levels were also lower in VHL tumours than in those where there was no hereditary syndrome. Relative absence of expression of NPY in phaeochromocytomas from VHL patients when compared with other groups appears to be largely independent of differences in catecholamine production and is consistent with a unique phenotype in VHL syndrome.

Background/methods: We have examined the hypothesis that cyst formation is key in the pathogenesis of cardiovascular disease in a Lewis polycystic kidney (LPK) model of autosomal-recessive polycystic kidney disease (ARPKD), by determining the relationship between cyst development and indices of renal function and cardiovascular disease., Results: In the LPK (n = 35), cysts appear at week 3 (1.1 +/- 0.1 mm) increasing to week 24 (2.8 +/- 2 mm). Immunostaining for nephron-specific segments indicate cysts develop predominantly from the collecting duct. Cyst formation preceded hypertension (160 +/- 22 vs. Lewis control 105 +/- 20 mm Hg systolic blood pressure (BP), n = 12) at week 6, elevated creatinine (109 +/- 63 vs. 59 +/- 6 micromol/l, n = 16) and cardiac mass (0.7 vs. 0.4% bodyweight, n = 15) at week 12, and left ventricular hypertrophy (2,898 +/- 207 vs. 1,808 +/- 192 mum, n = 14) at week 24 (all p < or = 0.05). Plasma-renin activity and angiotensin II were reduced in 10- to 12-week LPK (2.2 +/- 2.9 vs. Lewis 11.9 +/- 4.9 ng/ml/h, and 25.0 +/- 19.1 vs. 94.9 +/- 64.4 pg/ml, respectively, n = 26, p < or = 0.05). Ganglionic blockade (hexamethonium 3.3 mg/kg) significantly reduced mean BP in the LPK (52 vs. Lewis 4%, n = 9, p < or = 0.05)., Conclusion: Cyst formation is a key event in the genesis of hypertension while the sympathetic nervous system is important in the maintenance of hypertension in this model of ARPKD., (Copyright 2007 S. Karger AG, Basel.)

Pheochromocytomas are rare neuroendocrine tumors of chromaffin cell origin that synthesize and secrete excess quantities of catecholamines and other vasoactive peptides. Pheochromocytomas also express the norepinephrine transporter (NET), a molecule that is used clinically as a means of incorporating radiolabelled substrates such as 131I-MIBG (iodo-metaiodobenzylguanidine) into pheochromocytoma tumor cells. This allows the diagnostic localization of these tumors and, more recently, 131I-MIBG has been used in trials in the treatment of pheochromocytoma, potentially giving rise to NET as a therapeutic target. However, because of varying levels or activities of the transporter, the ability of 131I-MIBG to be consistently incorporated into tumor cells is limited, and therefore various strategies to increase NET functional activity are being investigated, including the use of traditional chemotherapeutic agents such as cisplatin or doxorubicin. Other aspects of NET discussed in this short review include the regulation of the transporter and how novel protein-protein interactions between NET and structures such as syntaxin 1A may hold the key to innovative ways to increase the therapeutic value of 131I-MIBG.

The sympathetic preganglionic neurons (SPN) of the intermediolateral cell column (IML) play a critical role in the maintenance of vascular tone. We undertook a comparative neuroanatomical analysis of neuronal nitric oxide synthase (nNOS) expression in the SPN of the mature normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rat (SHR). The anatomical relationship between nNOS and the NO signaling molecule cyclic guanosine monophosphate (cGMP) was also determined. All animals were male, age > 6 months. Fluorogold (FG) retrograde labeling of SPN (detected with immunohistochemistry) was combined with NADPH-diaphorase histochemistry for NOS in the thoracic spinal cord (T1-11, n = 5 WKY, 5 SHR). There was no difference in the total number of FG-labeled SPN (WKY 6,542 +/- 828, SHR 6,091 +/- 820), but the proportion of FG-labeled cells expressing NOS was significantly less in the SHR (WKY 64.4 +/- 5.1 vs. SHR 55.6 +/- 2.1, P < 0.05). Fluorescence immunohistochemistry for nNOS/cGMP (n = 4 WKY, 4 SHR) was also performed. Confocal microscopy revealed that all nNOS-positive SPN contain cGMP and confirmed a strain-specific anatomical arrangement of SPN cell clusters. A novel subpopulation of cGMP-only cells were also identified. Double labeling for cGMP and choline acetyltransferase (n = 3 WKY, 3 SHR), confirmed these cells as SPN in both WKY and SHR. These results suggest that cGMP is a key signaling molecule in SPN, and that a reduced number of NOS neurons in the SHR may play a role in the increase in sympathetic tone associated with hypertension in these animals., (Copyright 2006 Wiley-Liss, Inc.)

1. Activation of angiotensin II AT1 receptors (AT1R) stimulates catecholamine systems within both central and peripheral tissues that are associated with blood pressure control. In the present study, we sought to determine whether the hypertensive phenotype of the spontaneously hypertensive rat (SHR) is associated with changes in AT1R gene expression and whether gene expression of downstream signalling molecules was coupled to catecholamine gene expression, both in key brainstem nuclei and in peripheral sites implicated in cardiovascular control. 2. Gene expression levels of AT1R, extracellular signal-regulated kinase (ERK) 1 and 2 and phosphatidylinositol 3-kinase (PI3-K) were quantified in Wistar-Kyoto (WKY) rats and SHR. Messenger RNA expression levels were quantified using real time reverse transcription-polymerase chain reaction. In addition, we investigated whether there was a relationship between gene expression and systolic blood pressure. 3. The gene expression levels of AT1R, ERK2 and PI3-K were significantly higher in the paraventricular nucleus of the hypothalamus (4.12-, 1.40- and 1.38-fold, respectively), rostral ventrolateral medulla (2.71-, 1.33- and 2.73-fold, respectively), spinal cord (30.5-, 2.72- and 1.53-fold, respectively), adrenal medulla (1.68-, 1.55- and 1.76-fold, respectively) and coeliac ganglion (1.39-, 1.35- and 1.12-fold, respectively) in SHR compared with WKY rats. There was no significant difference in the level of ERK1 gene expression between the two strains. The gene expression levels of AT1R and ERK2 were positively correlated with blood pressure in all central nervous tissues investigated in the SHR, but not in WKY rats. Gene expression levels of the AT1R in the coeliac ganglion and adrenal medulla were also positively correlated with increased systolic blood pressure. 4. The present data suggest that a defect in AT1R expression (that may further alter downstream signalling pathways) in the SHR may be responsible, at least in part, for the hypertensive phenotype.

We have compared the expression pattern of NMDA receptor subunits (NR1 and NR2A-D) and NR1 splice variants (NR1-1a/1b,-2a/2b,-3a/3b,-4a/4b) in motor neuron populations from adult Wistar rats that are vulnerable (hypoglossal, XII) or resistant (oculomotor, III) to death in amyotrophic lateral sclerosis (ALS). The major finding was higher levels of expression of the NR2B subunit in the hypoglossal nucleus. Quantitative real-time PCR showed that NR1 was expressed at a greater level than any of the NR2 subunits (>15 fold greater, P

TLX1/HOX11, a DNA-binding homeodomain protein, was originally identified by virtue of its aberrant expression in T-cell leukemia and subsequently found to be crucial for normal spleen development. The precise mechanism of TLX1 function remains poorly understood, although it is known that it can act as both a transcriptional activator and repressor and can downregulate the Aldh1a1 gene in embryonic mouse spleen. Using a whole-genome PCR approach, we show here that TLX1 protein directly interacts with pericentromeric human satellite 2 DNA sequences. Such DNA is known to localize to heterochromatin, which among other roles has been implicated in gene silencing. The interaction was confirmed in vitro and in vivo by gel retardation and chromatin immunoprecipitation assays involving satellite 2 DNA, which contained sequences resembling TLX1 binding sites. Using immunofluorescence microscopy, TLX1 demonstrated a punctate pattern of staining in the nuclei of leukemic T-cells (ALL-SIL). Double labelling indicated that TLX1 colocalized with the centromeric protein CENP-B, demonstrating that the TLX1 foci corresponded to clusters of centromeric DNA. The novel interaction of TLX1 with constitutive heterochromatin adds an additional level of complexity to the intracellular functions of this transcriptional regulator and may have relevance to its roles in transcriptional repression and T-cell immortalization.

Expression of the noradrenaline transporter (NAT) was examined in normal human adrenal medulla and phaeochromocytoma by using immunohistochemistry and confocal microscopy. The enzymes tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were used as catecholamine biosynthetic markers and chromogranin A (CGA) as a marker for secretory granules. Catecholamine content was measured by using high performance liquid chromatography (HPLC). In normal human adrenal medulla (n=5), all chromaffin cells demonstrated strong TH, PNMT and NAT immunoreactivity. NAT was co-localized with PNMT and was located within the cytoplasm with a punctate appearance. Human phaeochromocytomas demonstrated strong TH expression (n=20 samples tested) but variable NAT and PNMT expression (n=24). NAT immunoreactivity ranged from absent (n=3) to weak (n=10) and strong (n=11) and, in some cases, occupied an apparent nuclear location. Unlike the expression seen in normal human adrenal medullary tissue, NAT expression was not consistently co-localized with PNMT. PNMT also showed highly variable expression that was poorly correlated with tumour adrenaline content. Immunoreactivity for CGA was colocalized with NAT within the cytoplasm of normal human chromaffin cells (n=4). This co-localization was not consistent in phaeochromocytoma tumour cells (n=7). The altered pattern of expression for both NAT and PNMT in phaeochromocytoma indicates a significant disruption in the regulation and possibly in the function of these proteins in adrenal medullary tumours.

We have compared PC12 cell lines derived from different laboratories and the newly developed mouse pheochromocytoma (MPC) cell line. Morphologically, there were distinct differences in size, shape, adherence, and clumping behavior, which varied in response to different culture media, growth substrates, and nerve growth factor. Quantitative messenger ribonucleic acid (mRNA) analysis showed significant variability in the expression of the catecholaminergic biosynthetic enzymes tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase (PNMT), the noradrenaline transporter (NAT), and neuron-specific enolase (NSE) between all lines examined. Of most significance were the increased levels of PNMT mRNA in the MPC cells, which were to 15-fold greater than in the PC12 cell lines grown under the same conditions in Dulbecco modified Eagle medium (P < or = 0.05). Growth of MPC cells in Roswell Park Memorial Institute media induced a further significant increase in PNMT gene expression (P < or = 0.05). Immunohistochemistry for TH, PNMT, and NAT was generally consistent with mRNA analysis, with the MPC cells demonstrating strong immunoreactivity for PNMT. The MPC cells showed the highest levels of desipramine-sensitive [(3)H] noradrenaline uptake activity (threefold > than PC12 American Type Culture Center line, P < or = 0.05), despite relatively low levels of NAT mRNA. These results indicate that PC12 cell lines should be carefully chosen for optimal utility in the study of chromaffin cell or sympathetic neuron biology and that cell features will be influenced by type of media and substrate chosen. Furthermore, they confirm that the new MPC cell line is likely a useful model for the study of adrenergic mechanisms or studies involving NAT.

1. The kidney receives a dense innervation of sympathetic and sensory fibres and can be both a target of sympathetic activity and a source of signals that drive sympathetic tone. In the normal state, interactions between the kidney and sympathetic nervous system (SNS) serve to maintain blood pressure and glomerular filtration rate within tightly controlled levels. In renal failure, a defect in renal sodium excretory function leads to an abnormal pressure natriuresis relationship and activation of the renin-angiotensin-aldosterone system, contributing to the development of hypertension and progression of kidney disease. 2. Evidence now strongly indicates a role for the SNS in the pathogenesis of hypertension in renal failure. Hypertension occurs commonly and early in renal disease and is paralleled by increases in SNS activity, as indicated by increased muscle sympathetic nerve activity and circulating catecholamines. This appears to be driven by the diseased kidneys, because nephrectomy or denervation has been shown to correct blood pressure and SNS activity in human and animal studies. 3. Afferent signals from the kidney, detected by chemoreceptors and mechanoreceptors, feed directly into central nuclei of the SNS, including the hypothalamus and circumventricular organs, in addition to the stimulus provided by circulating and brain-derived angiotensin II. Therefore, the pathogenesis of hypertension in renal failure is complex and arises from the interaction of haemodynamic and neuroendocrine factors. 4. Increased SNS activity has significant implications with regard to increased risk of cardiovascular disease and is an important consideration in the treatment of renal failure.

Distinct chemical codes are thought to reflect functional specificity in sympathetic preganglionic neurons (SPN). Although a number of chemical candidates have been identified including neurotransmitter-related, calcium-binding and other proteins, signal transduction proteins have been largely neglected. Not only might these chemicals allow discrimination of functionally unique chemical signatures, but they may also identify activated neurons. Immunoreactivity (ir) to phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was differentially located within the thoracic spinal cord depending upon which of three forms of killing was used: the only exception to this was the intermediolateral cell column (IML) which was consistently, densely labeled. The presence or absence of p-ERK1/2 in SPN (n=17,541) within the IML of the thoraco-lumbar spinal cord was determined in seven rats. SPN were identified on the basis of their location, size and that they contained choline acetyltransferase ir. On average, 58% of SPN contained p-ERK1/2, however, more SPN in both the upper (72%; C8-T4) and lower (78%; T11-L3) thoraco-lumbar spinal cord contained p-ERK1/2-ir than the middle thoracic region (47%; T4-T10). p-ERK1/2-ir was also examined in SPN (n=1895) innervating the adrenal medulla (identified by retrograde tracing using cholera toxin B subunit) combined with localization of neuronal nitric oxide synthase (nNOS) in three rats. On average, 64% of adrenal SPN contain p-ERK1/2-ir, and it was confirmed that all adrenal SPN contain nNOS-ir. It appears that p-ERK1/2-ir SPN, described in this study, have tonically activated receptors that are coupled to intracellular signal transduction pathways that lead to the phosphorylation of ERK1/2.

The rostral ventrolateral medulla (RVLM) is the major brainstem region contributing to sympathetic control of blood pressure. We have compared the expression of N-methyl-d-aspartate (NMDA) receptor subunits (NR1, NR2A-D), NR1 splice variants (NR1-1a/1b, -2a/2b, -3a/3b, -4a/4b), and the neuronal and inducible isoforms of NO synthase (nNOS and iNOS) in the RVLM of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR), based on the hypothesis that altered NMDA receptor make-up or altered expression of endogenous NO may be associated with the increase in sympathetic output described from this site in hypertension. Total RNA was extracted and reverse transcribed from the RVLM of mature male WKY and SHR (16-23 weeks). Conventional polymerase chain reaction (PCR) indicated that only the NR1 splice variants NR1-2a, NR1-2b, NR1-4a and NR1-4b were expressed in the RVLM of either species. Quantitative real-time PCR indicated that for both strains of rat, mRNA for the NR1 subunit (all splice variants) was the most abundant (16.5-fold greater, P< or =0.05, relative to the NR2A subunit). Amongst the NR2A-D subunits, NR2C was the most abundant (7- and 1.7-fold greater relative to the NR2A subunit, P< or =0.05, WKY and SHR, respectively). Relative to WKY, mRNA levels for the NR2C and NR2D subunits in the SHR RVLM were significantly lower (0.3- and 0.25-fold less, P< or =0.05), while nNOS was significantly higher (1.76-fold greater, P< or =0.05). This was confirmed immunohistochemically for nNOS expression. These results demonstrate differential expression levels of NMDA receptor subunits and NOS isoforms in the RVLM region of SHR when compared to WKY rats.

On December 2, 1999, 120 pregnant cows were weighed, their body condition scored, and then sorted into six groups of 20 stratified by BCS, BW, breed, and age. Groups were assigned randomly to six, 5.1-ha dormant common bermudagrass (Cynodon dactylon [L.] Pers.) pastures for 2 yr to determine the effects of supplemental Se and its source on performance and blood measurements. During the winter, each group of cows had ad libitum access to bermudagrass/dallisgrass (Paspalum dilatatum Poir.) hay plus they were allowed limited access (1 to 4 d/wk) to a 2.4-ha winter-annual paddock planted in half the pasture. Treatments were assigned randomly to pastures (two pastures per treatment), and cows had ad libitum access to one of three free-choice minerals: 1) no supplemental Se, 2) 26 mg of supplemental Se from sodium selenite/kg, and 3) 26 mg of supplemental Se from seleno-yeast/kg (designed intake = 113 g/cow daily). Data were analyzed using a mixed model; year was the random effect and treatment was the fixed effect. Selenium supplementation or its source had no effect (P > or = 0.19) on cow BW, BCS, conception rate, postpartum interval, or hay DMI. Birth date, birth weight, BW, total BW gain, mortality, and ADG of calves were not affected (P > 0.20) by Se or its source. Whole blood Se concentrations and glutathione peroxidase (GSH-Px) activity at the beginning of the trial did not differ (P > or = 0.17) between cows receiving no Se and cows supplemented with Se or between Se sources. At the beginning of the calving and breeding seasons, cows supplemented with Se had greater (P < 0.01) whole blood Se concentrations and GSH-Px activities than cows receiving no supplemental Se; cows fed selenoyeast had greater (P < or = 0.05) whole blood Se concentrations than cows fed sodium selenite, but GSH-Px did not differ (P > or = 0.60) between the two sources. At birth and on May 24 (near peak lactation), calves from cows supplemented with Se had greater (P < or = 0.06) whole blood Se concentrations than calves from cows fed no Se. At birth, calves from cows fed seleno-yeast had greater (P < or = 0.05) whole blood Se concentrations and GSH-Px activities than calves from cows fed sodium selenite. Although no differences were noted in cow and calf performance, significant increases were noted in whole blood Se concentrations and GSH-Px activities in calves at birth as a result of feeding of seleno-yeast compared to no Se or sodium selenite.

Introduction: Prone positioning in respiratory failure has been shown to be a useful adjunct in the treatment of severe hypoxia. However, the prone position can result in dislodgment or malfunction of tubes and cannulae. Certain patients receiving extracorporeal membrane oxygenation (ECMO) or continuous renal replacement therapy (CRRT) may also benefit from positional therapy. The impact of cannula-related complications in these patients is potentially disastrous. The safety and efficacy of prone positioning of these patients has not been previously reported., Materials and Methods: A retrospective chart review evaluated ECMO or CRRT cannula location, and displacement or malfunction during positional change or while prone. The study was set in a General Surgery and Trauma Intensive Care Unit. The subjects were all patients at our institution who simultaneously underwent ECMO or CRRT and prone positioning from July 1996 to July 2001. There were no interventions., Results: Ten patients underwent ECMO and 42 patients underwent CRRT during the study period. Seven patients underwent simultaneous prone positioning and either ECMO (4/10) or CRRT (4/42). A total of 68 turning events (prone to supine or supine to prone) were recorded, with each patient averaging 9.7 (range, 4-16) turning episodes. Turning was performed with sheets and extra nursing personnel; no special mechanical assist devices were used. No patients experienced inadvertent cannula removal during turning. Two patients had poor flow through their cannulae. In one patient, this occurred in the supine position and required repositioning of the cannula. In the second patient, cannulae were changed twice and flow was poor in both the supine and the prone positions. All ECMO and CRRT patients received venous cannulae. Cannula location (seven internal jugular and 11 femoral) did not the affect risk of malfunction., Discussion and Conclusions: Patients with venous cannulae for ECMO or CRRT can be safely placed in the prone position. Flow rates are maintained in this position. Potential cannula complications of ECMO and CRRT are not a contraindication to prone positioning in severely ill patients.

The aims of this study were, first, to determine quantitatively the levels of tyrosine hydroxylase (TH) gene expression in both peripheral and central sites related to blood pressure regulation, and to compare the level of expression in Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Second, to see if any relationship exists between TH gene expression and systolic arterial blood pressure. Total RNA was isolated from adrenal glands and from tissue punches taken from the C1 and A1 cell groups in the rostral and caudal ventrolateral medulla oblongata of the brainstem, respectively. Total RNA was reverse-transcribed into cDNA followed by quantitative fluorescence detection polymerase chain reaction for TH cDNA. The levels of TH gene expression measured as a percentage of the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in SHR, were significantly higher ( approximately 2.5-fold) compared to WKY in all sites examined (P<0.01). There was a positive and significant relationship between systolic blood pressure and TH gene expression in the C1 area of the brainstem in both WKY (n = 5, P<0.05) and SHR (n=6, P<0.05). Taken together, these results suggest that elevated gene expression of the TH gene is associated with the phenotypic characteristic of SHR.

It has been demonstrated that arginine vasopressin (AVP) is synthesized not only in specific hypothalamic nuclei, but also in the adrenal medulla where it is thought to regulate adrenal functions by autocrine and paracrine mechanisms. In order to further characterise the effects of AVP on rat adrenal chromaffin cells, we examined: (a) the mRNA expression for V(1a) and V(1b) AVP receptors in these cells; (b) the effects of AVP on the membrane potential and membrane currents measured with the whole-cell patch-clamp technique; and (c) effect of AVP on catecholamine release from single adrenal chromaffin cells measured with carbon fibre microelectrodes. Reverse transcription-polymerase chain reaction (RT-PCR) on tissue punch samples obtained from the adrenal medulla demonstrated message for both the V(1a) and V(1b) receptors, while material obtained from the adrenal cortex showed expression of the V(1a) receptor only. Single-cell RT-PCR conducted on acutely isolated chromaffin cells showed message for the V(1a) receptor in 84% of cells, while 38% of cells also contained message for the V(1b) receptor (n=45). Under current-clamp recording, responses to AVP application (4-40 microM) were variable; 22/34 (65%) tested cells were depolarised, 29% hyperpolarised, and the remaining cells showed a biphasic response. Changes in membrane potential of either direction were dose-dependent and accompanied by a decrease in cell membrane resistance. Under voltage-clamp (V(hold)=-60 mV), AVP evoked inward current in 27/52 (52%) and outward current in 16/52 (31%) chromaffin cells. Both types of AVP-evoked responses were blocked by co-application of a nonselective V(1a)/V(1b) antagonist. Application of AVP evoked prolonged bursts of amperometric currents (indicative of catecholamine release) in 4/9 tested cells, but reduced the currents evoked by ACh application in all tested cells (n=7). These findings demonstrate a complex action of AVP on adrenal chromaffin cells, with individual adrenal chromaffin cells responding with either excitation or inhibition. This response pattern may be related to the expression of V(1) receptor subtypes.

The analysis of colocalization of multiple catecholamine biosynthetic enzymes within the ventrolateral part of the medulla oblongata of the rat revealed distinct subpopulations of neurons within the C1 region (Phillips et al., J Comp Neurol 2001, 432:20-34). In extending this study to include the caudal pons, it was shown for the first time that the A5 cell group could be distinguished by the presence of immunoreactivity to tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase (AADC), and dopamine beta hydroxylase (DBH). A novel cell group was also identified. The cells within this new group were immunoreactive to DBH but not TH, AADC, or phenylethanolamine N-methyltransferase (PNMT) and will be referred to as the TH-, DBH+ cell group. The TH-, DBH+ neurons were not immunoreactive for either the dopamine or noradrenaline transporters, suggesting that these neurons do not take up these transmitters. A5 neurons were immunoreactive for the noradrenaline transporter but not the dopamine transporter (as previously shown). Retrograde tracing with cholera toxin B revealed that the TH-, DBH+ neurons do not project to the thoracic spinal cord or to the rostral ventrolateral medulla, but A5 neurons do. A calbindin immunoreactive cell group is located in a region overlapping TH-, DBH+ cell group. However, only a few neurons were immunoreactive for both markers. The physiological role of the TH-, DBH+ cell group remains to be determined., (Copyright 2001 Wiley-Liss, Inc.)

The aim of this study was to investigate the expression of the alpha2-adrenergic receptors in the adrenal medulla, and to examine the mechanism by which clonidine and related drugs inhibit acetylcholine (ACh)-induced whole-cell currents in adrenal chromaffin cells. Reverse transcription-polymerase chain reaction (RT-PCR) performed on punches of rat adrenal medulla demonstrated expression of mRNA for the 2A-, alpha2B- and alpha2C-adrenergic receptors. Similar experiments conducted with tissue punches obtained from the adrenal cortex did not reveal expression of these receptor subtypes. Whole-cell currents were recorded in isolated chromaffin cells using the perforated-patch configuration. ACh (50 microM) evoked inward currents with a peak amplitude of 117.8+/-9.3 pA (n = 45; Vhol = -60 mV). The currents were inhibited in a dose-dependent manner (0.5-50 microM) by clonidine, UK 14,304 and rilmenidine (agonists of alpha2/imidazoline receptors), as well as by SKF 86466 and efaroxan (antagonists). Adrenaline and noradrenaline (50-100 microM) had no significant effect. Thus, although the adrenal medulla expresses mRNA for the alpha2-adrenergic receptors, the lack of agonist-antagonist specificity observed in our whole-cell recordings (in the absence of intracellular dialysis) provides additional evidence against the possibility that these inhibitory effects are mediated by classical alpha2 or imidazoline receptor interactions.

Adrenergic (C1) neurons located in the rostral ventrolateral medulla are considered a key component in the control of arterial blood pressure. Classically, C1 cells have been identified by their immunoreactivity for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and/or phenylethanolamine N-methyltransferase (PNMT). However, no studies have simultaneously demonstrated the expression of aromatic L-amino acid decarboxylase (AADC) and dopamine beta-hydroxylase (DBH) in these neurons. We examined the expression and colocalization of all four enzymes in the rat ventrolateral medulla using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Retrograde tracer injected into thoracic spinal segments T2-T4 was used to identify bulbospinal neurons. Using fluorescence and confocal microscopy, most cells of the C1 group were shown to be double or triple labeled with TH, DBH, and PNMT, whereas only 65-78% were immunoreactive for AADC. Cells that lacked detectable immunoreactivity for AADC were located in the rostral C1 region, and approximately 50% were spinally projecting. Some cells in this area lacked DBH immunoreactivity (6.5-8.3%) but were positive for TH and/or PNMT. Small numbers of cells were immunoreactive for only one of the four enzymes. Numerous fibres that were immunoreactive for DBH but not for TH or PNMT were noted in the rostral C1 region. Single-cell RT-PCR analysis conducted on spinally projecting C1 neurons indicated that only 76.5% of cells that contained mRNA for TH, DBH, and PNMT contained detectable message for AADC. These experiments suggest that a proportion of C1 cells may not express all of the enzymes necessary for adrenaline synthesis., (Copyright 2001 Wiley-Liss, Inc.)

Expression of the noradrenaline transporter (NAT) was identified in various cell and fibre populations of the rat adrenal medulla, examined with immunohistochemistry and confocal microscopy. Immunoreactivity for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), aromatic-L-amino-acid decarboxylase (AADC) and dopamine beta-hydroxylase (DBH) was present in all chromaffin cells, while phenylethanolamine N-methyltransferase (PNMT) was used to determine adrenergic chromaffin cell groups. Labelling with NAT antibody was predominantly cytoplasmic and colocalised with PNMT immunoreactivity. Noradrenergic chromaffin cells were not NAT immunoreactive. Additionally, NAT antibody labelling demonstrated clusters of ganglion cells (presumably Type I) and nerve fibres. Expression of TH, AADC, DBH, PNMT and NAT mRNA was examined using reverse transcription-polymerase chain reaction (RT-PCR) from adrenal medulla punches and single chromaffin cells, and results were consistent with those obtained with immunocytochemistry. Chromaffin cells and fibres labelled with antibodies against growth associated protein-43 (GAP-43) were not NAT immunoreactive, while ganglion cells were doubled labelled with the two antibodies. The presence of NAT in adrenergic chromaffin cells, and its absence from noradrenergic cells, suggests that the adrenergic cell type is primarily responsible for uptake of catecholamines in the adrenal medulla.

The control and maintenance of vascular tone is due to a balance between vasoconstrictor and vasodilator pathways. Vasomotor responses to neural, metabolic and physical factors vary between vessels in different vascular beds, as well as along the same bed, particularly as vessels become smaller. These differences result from variation in the composition of neurotransmitters released by perivascular nerves, variation in the array and activation of receptor subtypes expressed in different vascular beds and variation in the signal transduction pathways activated in either the vascular smooth muscle or endothelial cells. As the study of vasomotor responses often requires pre-existing tone, some of the reported heterogeneity in the relative contributions of different vasodilator mechanisms may be compounded by different experimental conditions. Biochemical variations, such as the expression of ion channels, connexin subtypes and other important components of second messenger cascades, have been documented in the smooth muscle and endothelial cells in different parts of the body. Anatomical variations, in the presence and prevalence of gap junctions between smooth muscle cells, between endothelial cells and at myoendothelial gap junctions, between the two cell layers, have also been described. These factors will contribute further to the heterogeneity in local and conducted responses.

In studies of the central and peripheral autonomic nervous system, it has become increasingly important to be able to investigate mRNA expression patterns within specific neuronal populations. Traditionally, the identification of mRNA species in discrete populations of cells has relied upon in situ hybridization. An alternative, relatively simple procedure is 'multiplex' reverse transcription-polymerase chain reaction (RT-PCR), conducted on single neurons after their in vitro isolation. Multiplex single-cell RT-PCR can be used to examine the expression of multiple genes within individual cells, and can be combined with electrophysiological, pharmacological and anatomical (retrograde labelling) studies. This review focuses on a number of key aspects of this approach, methodology, and both the advantages and the limitations of the technique. We also provide specific examples of work performed in our laboratory, examining the expression of alpha 2-adrenergic receptors in catecholaminergic cells of the rat brainstem and adrenal medulla. The application of single-cell RT-PCR to future studies of the autonomic nervous system will hopefully provide information on how physiological and pathological conditions affect gene expression in autonomic neurones.

We have characterised nerve-mediated vasodilations in small arteries of the rat hepatic mesentery. Stimulation of sympathetic nerves (10 Hz, 10 s) produced a vasodilation which was abolished by the beta-adrenoceptor antagonist, propranolol (2 x 10(-6) M), but was unaffected by NG-nitro-L-arginine methyl ester (L-NAME, 10(-5) M). Stimulation of sensory nerves produced a large vasodilation that was abolished by capsaicin (10(-6) M). This vasodilation was unaffected by L-NAME (10(-5) M), but significantly reduced by the calcitonin gene related peptide (CGRP) antagonist, CGRP8-37 (10(-6) M), or inhibition of adenylate cyclase (SQ22356, 2 x 10(-5) M; 2',5'-dideoxyadenosine, 2 x 10(-4) M). Stimulation of cholinergic nerves produced a small vasodilation which was significantly reduced by scopolamine (10(-6) M). Expression of mRNA for CGRP1 receptors, muscarinic m2, m3 and m5 receptors and neurokinin1 (NK1) and NK3, receptors was detected. Perivascular nerves were immunoreactive for CGRP and substance P. No role was found for substance P, neuronal NO, ATP or adenosine in nerve-mediated responses. L-NAME (10(-5) M) potentiated vasoconstrictions following sympathetic nerve stimulation. This effect was reversed by L-arginine (10(-3) M) and cromakalim (10(-6) M) and mimicked by glybenclamide (10(-5) M), thus implicating KATP channels. Vasodilation in response to sensory nerve stimulation was directly proportional to the level of preconstriction, while vasodilation in response to neurogenic or applied acetylcholine was inhibited at high levels of preconstriction. We hypothesize that, under conditions of intensive vasoconstriction, some endothelial-dependent vasodilations may be less important than vasodilations activated directly through the smooth muscle.

1. The functional innervation of autonomic target tissues occurs early during development, at a time when both the nerves and post-synaptic target tissues are still differentiating. 2. Physiological responses appear soon after the arrival of the first fibres when uptake and release mechanisms within the nerves are already functional. Initial responses differ from those in the mature animal, both in the form and, frequently, in the subtypes of receptors involved. 3. Results of a number of studies suggest that the initial expression of neurotransmitter receptors during development is largely independent of neural influences. Changes recorded in neurotransmitter receptor expression during development appear to be similarly independent of neural influences. 4. While signal transduction pathways coupling adrenergic neurotransmitter receptors to effector responses appear to develop independently of the nerves, the efficient coupling of muscarinic receptors often requires the action of the neurotransmitter, acetylcholine. 5. During the period of synapse formation, the neural plexus continues to expand. While developing varicosities can release the neurotransmitter, the capacity for neurotransmitter retention appears to be restricted. Developmental changes in the neurotransmitters that produce functional responses, while well known in the sweat glands, may also be seen in more subtle forms in other target tissues. 6. Ultrastructural studies suggest that close physical associations between the membranes of the release sites of the developing nerves and the target cells may form early during development when physiological responses are still immature. These close associations could enable more specific reciprocal interactions between nerves and target cells involving known and novel growth factors, neuropeptides and cytokines important in shaping the mature synaptic characteristics.

Studies in autonomic targets have shown that nerves may be required for the development and maintenance of postsynaptic receptor populations. We have examined this relationship in the rat mesenteric artery, assessing mRNA expression levels for a range of neuroreceptors after neonatal sympathectomy, using 6-hydroxydopamine or antisera directed against nerve growth factor, and sensory denervation, using capsaicin. Total RNA was extracted from 28 day old rats and reverse transcription-polymerase chain reaction was performed, using primers specific for the alpha1(A,B,D)- and alpha2(A,B,C)-adrenergic, neurokinin (NKI-NK3), muscarinic (M1-M5) and P2X purinergic (P2x1-7) receptor families. Results showed no decreases in mRNA expression of any of the specific receptor subtypes after either sympathetic or sensory denervation. Small increases in mRNA expression were detected following sensory denervation for some of the receptor subtypes. We conclude that neither sympathetic nor sensory nerves are mandatory for the expression of mRNA of a range of neuroreceptors in the mesenteric vascular bed of the rat.

Acute respiratory distress syndrome is a complex clinical syndrome of respiratory failure that presents a challenge to every critical care team. Since the first clear description by Ashbaugh et al more than 30 years ago, much has been learned about the pathophysiologic process that occurs within the lungs after they suffer either a direct or indirect injury. Unfortunately, little success has been achieved in improving outcomes; however, hope is on the horizon. Current research evaluating optimal ventilator management, ECMO, the use of inhaled nitric oxide, and other experimental management strategies will hopefully combine to produce improved outcomes.

1. We have investigated the neurotransmitters and receptor subtypes involved in nerve-mediated vasoconstriction in small arteries of the rat hepatic mesentery. 2. A dense sympathetic innervation was demonstrated using catecholamine histochemistry and antibodies against the synaptic vesicle protein synaptophysin. 3. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated very strong expression of the alpha1A-adrenergic, neuropeptide Y (NPY) Y1, P2X1- and P2X4-purinergic receptors, moderate expression of the alpha2B-adrenergic receptor and the purinergic P2X5- and P2X7-receptors and weak expression of the alpha1B-, alpha1D-, alpha2A- and alpha2C-adrenergic receptors and the P2X2- and P2X3-purinergic receptors. NPY2 and P2X6 receptor expression was absent. 4. Electrical field stimulation (10 Hz, 10 s) produced contractions which were abolished by tetrodotoxin (10(-6) M) and/or guanethidine (GE, 5 x 10(-6) M) and a combination of benextramine (10(-5) M) and alpha,beta-methylene ATP, (alpha,beta-mATP, 3 x 10(-6) M) or PPADS (10(-5) M). Selective alpah1-adrenergic receptor antagonists showed the potency order of prazosin > WB-4101 > 5-methyl-urapidil > BMY 7378. Yohimbine (10(-8) M, 10(-7) M), alpha,beta-mATP (3 x 10(-6) M) and PPADS (10(-5) M) each enhanced the response to nerve stimulation. 5. Some experiments demonstrated a slow neurogenic contraction which was abolished by GE or the selective NPY1 receptor antagonist 1229U91 (6 x 10(-7) M). 6. We conclude that nerve-mediated vasoconstriction results from the activation of postsynaptic alpha,beta-adrenergic and P2X-purinergic receptors and under some conditions, NPY1 receptors. Neurotransmitter release is modulated by presynaptic alpha2-adrenergic receptors and possibly also P2X-purinoceptors. The major postsynaptic subtypes involved were well predicted by mRNA expression as measured by RT-PCR, suggesting that this technique may be a useful adjunct to studies aimed at identifying functional receptor subtypes.

Different mechanisms mediate constriction and dilation in different vascular beds. We have used reverse transcription-polymerase chain reaction to investigate whether specific patterns of receptor gene expression may underlie these variable responses. Total RNA, from the basilar, pulmonary, mesenteric and tail arteries of anaesthetised adult Wistar rats, was reverse transcribed and amplified using primers specific for the molecular subtypes of the alpha 1(A, B, D)- and alpha 2(A, B, C)-adrenergic, neurokinin (NK1-NK3) and muscarinic (m1-m5), receptors. Results showed that the pattern of gene expression was variable with no two arteries having the same receptor profile. Messenger RNA for the alpha 1A, alpha 1B, alpha 2B, NK1, NK3, m3 and m5 receptor subtypes were detected in all vessels studied while the remaining subtypes showed a variable expression amongst the arteries. This is the first description of mRNA for the m5 muscarinic receptor in peripheral tissue. The NK3 receptor was the major neurokinin receptor expressed in all vessels except the pulmonary artery, in which the NK1 receptor was also strongly expressed. We conclude that each artery expressed a specific receptor array which may permit some unique neural and hormonal controls.

Developmental studies show that the innervation of autonomic targets is accompanied by increases in the density of receptors, maturation of receptor-signalling pathways and changes in receptor subtype. The innervation of the rat mesenteric artery occurs over the first 3 postnatal weeks. In this study, we investigated whether alterations in receptor gene expression may underlie physiological changes recorded during development and maturity in this vessel. Total RNA, from mesenteric arteries of rats at birth and postnatal days 7, 14, 28, 240 and 360, was reverse transcribed and amplified using primers specific for the alpha 1 (A, B, D)- and alpha 2 (A, B, C)-adrenergic, neurokinin (NK1-NK3) and muscarinic (m1-m5) receptors. Results showed that all receptor genes expressed at 28 days, except the alpha 1D-adrenergic receptor, were already expressed at birth. Some receptor subtypes showed no change in their relative expression, always being either strongly (alpha 1A, alpha 2B, NK3) or weakly (alpha 2A, alpha 2C, NK1) expressed. Relative to the expression of these receptors, others showed a developmental increase in expression up to 14 days postnatal (alpha 1B, alpha 1D, m2, m3, m5) but no further change with maturity. These latter changes coincide with the development of sympathetic and sensory nerve plexuses in the mesenteric artery, but do not correlate with the physiological changes seen during development and ageing.

Female cowpea weevils,Callosobruchus maculatus, produce a sex pheromone that elicits orientation and sexual behavior in males. Bioassay-directed isolation of the sex pheromone was conducted and compounds in the active fraction were identified and synthesized. Volatiles were collected from individual virgin females by adsorption on filter paper dises and hexane extraction. A bioassay was used in which the locomotory response of single males in glass vials was recorded upon exposure to treatments or controls. Crude extracts were subjected to silica gel column chromatography with solvents of increasing polarity; all activity eluted with methanol. Activity in the highly polar methanol fraction suggested a carboxylic acid or a compound with multiple polar functionality. Acid-base partitioning of the crude extract isolated all activity in the acid fraction, confirming that the pheromone was a carboxylic acid. The acid fraction was further fractionated by preparative GC with a Carbowax column. The most active GC fraction contained the following five 8-carbon acids identified by GC-MS and comparison with synthetic candidates: 3-methyleneheptanoic acid, (Z)-3-methyl-3-heptenoic acid, (E)-3-methyl-3-heptenoic acid, (Z)-3-methyl-2-heptenoic acid, and (E)-3-methyl-2-heptenoic acid. Each of the synthetic acids was active individually for males, and combinations of two or more of the acid pheromones had an additive effect. Upwind flight responses to natural and synthetic pheromones were observed in a flight tunnel. (Z)-3-Methyl-2-heptenoic acid was previously identified as the sex pheromone for the relatedC. analis, but this and the other four acid pheromones fromC. maculatus were inactive for maleC. analis. There was no cross-attraction betweenC. maculatus andC. analis in reciprocal studies using extracted volatiles from females of both species, GC-MS analysis ofC. analis female volatiles failed to detect any of theC. maculatus compounds but did find an unidentified C-8 acid with a GC retention time different from any of theC. maculatus pheromones.

Carbon tetrachloride (CCl4) lethality in Sprague-Dawley rats is greatly amplified by pretreatment of Kepone (decachlorooctahydro-1,3,2-metheno-2H-cyclobuta[cd] pentalen-2-one). The increase in lethality was attributed to the obstruction of liver regenerative processes. These processes are essential for restoring the liver to its full functional capacity following injury by CCl4. Based on the available mechanistic information on Kepone/CCl4 interaction, a physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) model was constructed where the following effects of Kepone on CCl4 toxicity are incorporated: (1) inhibition of mitosis; (2) reduction of repair mechanism of hepatocellular injury; (3) suppression of phagocytosis. The PBPK/PD model provided computer simulation consistent with previously published time-course results of hepatotoxicity (i.e., pyknotic, injured and mitotic cells) of CCl4 with or without Kepone. As a further verification of this model, the computer simulations were also consistent with exhalation kinetic data for rats injected with different intraperitoneal (i.p.) doses of CCl4 in our laboratory. Subsequently, the PBPK/PD model, coupled with Monte Carlo simulation, was used to predict lethalities of rats treated with CCl4 alone and CCl4 in combination with Kepone. The experimental lethality studies performed in our laboratories were as follows: Sprague-Dawley rats were given either control diet or diet containing 10 ppm Kepone for 15 days. On day 16, rats in the Kepone treated group were given i.p. doses of 0, 10, 50, and 100 microliters/kg CCl4 (n = 9) while control rats were exposed to 0, 100, 1000, 3000, and 6000 microliters/kg CCl4 (n = 9). Lethality was observed at the 1000 (1/9), 3000 (4/9), and 6000 (8/9) microliters/kg doses for the control group and at the 50 (4/9) and 100 (8/9) microliters/kg for the treated group. Based on Monte Carlo simulation, which was used to run electronically 1000 lethality experiments for each dosing situation, the LD50 estimates for CCl4 toxicity with and without Kepone pretreatment were 47 and 2890 microliters/kg, respectively. Monte Carlo simulation coupled with the PBPK/PD model produced lethality rates which were not significantly different from the observed mortality, with the exception of CCl4 at very high doses (e.g., 6000 microliters/kg, p = 0.014). Deviation at very high doses of the predicted mortality from the observed may be attributed to extrahepatic systemic toxicities of CCl4, or solvent effects on tissues at high concentrations, which were not presently included in the model. Our modeling and experimental results verified the earlier findings of Mehendale (1990) for the 67-fold amplification of CCl4 lethality in the presence of Kepone. However, much of this amplification of CCl4 lethality with Kepone pretreatment was probably due to pharmacokinetic factors, because when target tissue dose (i.e., model estimated amount of CCl4 metabolites) was used to evaluate lethality, this amplification was reduced to 4-fold.

57 patients with newly diagnosed intermediate or high grade non-Hodgkin's lymphoma with stage II to IV disease were treated with P-COMM-B (prednisolone, cyclophosphamide, vincristine, mitozantrone, methotrexate and bleomycin). 46% patients achieved a complete remission and 26% achieved a partial remission. Projected disease-free survival in complete remission at 5 years is 56% and projected overall survival at 5 years is 37%. Neutropenia and proximal myopathy were the commonest severe toxicities encountered and two deaths were clearly related to treatment (3.5%). P-COMM-B is effective first-line chemotherapy in intermediate and high grade non-Hodgkin's lymphoma. The efficacy and toxicity of P-COMM-B appear to be comparable to those of the best contemporary regimen, CHOP.

67 patients with relapsed or resistant multiple myeloma were randomized to receive either VAD (vincristine, doxorubicin, dexamethasone) or MOD (mitozantrone, vincristine, dexamethasone). 12/30 (40%) patients receiving VAD and 15/37 (41%) patients receiving MOD achieved plateaux. The median duration of plateaux was significantly longer on VAD (15 months) than on MOD (8 months). No significant difference in overall survival was seen between the two treatment arms. The only toxicity which was severe in more than 5% of treatment cycles on either treatment arm was myelosuppression. No toxicity was significantly more severe on MOD than VAD. However, hair loss was significantly more severe on VAD than MOD. The frequencies of thrombocytopenia, haematuria and cutaneous toxicity were significantly greater on VAD than on MOD. Raised serum direct bilirubin levels were seen significantly more often on MOD than VAD. MOD and VAD have similar efficacy in relapsed/resistant multiple myeloma. MOD is the less toxic of the two regimens.

A case of non-fatal massive bone marrow necrosis (MBMN) is reported in a patient with primary thrombocythaemia five years after diagnosis. No precipitating cause was identified. The patient developed postnecrotic myelofibrosis six months later. As far as is known, this is the first report of MBMN in a case of primary thrombocythaemia.

A case of B-CLL which was complicated by chronic renal failure due to leukaemic infiltration of the kidney is reported. Treatment with chlorambucil, prednisolone, and renal bed irradiation resulted in a substantial improvement in renal function which persisted until the the patient's death from marrow failure some eight years later. The temporal association between treatments and response suggested that renal bed radiotherapy had contributed to the improvement in renal function. This case is one of only two reported cases in which chronic renal failure due to CLL has been treated with radiotherapy. It is unique in that the renal response was shown histologically. Leukaemic infiltration of the kidney is common in CLL but, characteristically, is not associated with renal impairment. An improvement in renal function has been described in two patients with acute renal failure after chemotherapy.

A patient with autosomal dominant (Type III) von Willebrand's disease (vWd) developed acquired haemophilia post-operatively, possibly due to exposure to amoxycillin. She refused porcine factor VIII (pFVIII) on religious grounds and was managed successfully with recombinant activated factor VII (rFVIIa) together with highly purified von Willebrand factor concentrate (vWf-VHP). In patients with acquired haemophilia rFVIIa appears to be a suitable agent for symptomatic management.

We report high-volume postobstructive choleresis in two patients who underwent transhepatic external drainage for malignant biliary obstruction. Excessive loss of bicarbonate-rich biliary fluid (up to 6.5 L/day) caused orthostatic hypotension, prerenal insufficiency, hyponatremia, and a decrease in serum bicarbonate. Therapy with isotonic fluids containing sodium, chloride, lactate, bicarbonate, and potassium was based on measurement of biliary fluid volume and electrolyte concentrations. Biliary fluid loss was terminated by conversion to internal biliary drainage. The reason for this rare complication of external drainage of biliary obstruction is unknown, but such patients must be closely monitored for volume loss. When high-volume choleresis occurs, biliary fluid and electrolyte losses should be precisely measured and replaced, and external biliary drainage converted to internal drainage.

Heparin-induced skin reaction is a recognized complication of subcutaneously administered, unfractionated heparin, and has recently been described in association with the use of two LMWH preparations, Fragmin and Fraxiparin. We describe the case of a 25-year-old woman in whom characteristic skin lesions were induced on separate occasions by unfractionated heparin, Fragmin and a third preparation of LMWH, Clexane. This case demonstrates that substitution of one preparation of heparin for another does not prevent recurrence of skin reaction in susceptible patients. Alternative anticoagulants should be considered in these cases.

Laboratory experiments were conducted to study the behavioral activity of grain-derived volatiles as attractants and pheromone synergists forSitophilus oryzae, an internal-feeding pest of sound grain, andTribolium castaneum, an external-feeding pest of damaged grains and flour. Behavioral studies with two-choice pitfall bioassays determined that the fresh grain volatiles valeraldehyde, maltol, and vanillin were attractive toS. oryzae at various doses, butT. castaneum were not attracted to any dose of any of these three compounds. When oils from pressed grains were bioassayed, sesame oil was significantly repellent and oat and wheat germ oils were attractive toS. oryzae. However, rice, soybean, oat, wheat germ, and corn oils were all attractive toT. castaneum. A commercial food product composed primarily of soybean oil and wheat germ was highly attractive toT. castaneum, but elicited no response fromS. oryzae. A combination of the three grain volatiles valeraldehyde, maltol, and vanillin with the synthetic pheromone sitophinone was more attractive toS. oryzae than either the pheromone alone or the tripartite grain volatile mix. Similarly, a combination of the commercial food product with the pheromone 4,8-dimethyldecanal was more attractive toT. castaneum than either food alone or pheromone alone. Behavioral responses to grain volatiles may reflect the ecological niche of the granivore:S. oryzae colonizes sound grain and is attracted to volatiles characteristic of fresh grain, whileT. castaneum utilizes damaged or deteriorated grains and responds best to oils characteristic of damaged or fungus-infested grain. Synergism of food odors and pheromones suggests that more effective traps can be devised for management of these pest insects.