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Chronic pressure overload (PO) is associated with cardiac hypertrophy and altered autonomic control of cardiac function, in which the latter may involve adaptations in central and/or peripheral cardiac neural control mechanisms. To evaluate the specific remodeling of the intrinsic cardiac nervous system following pressure overload, the descending thoracic aorta artery of the guinea pig was constricted ~20%, and the animals recovered for 9 wk. Thereafter, atrial neurons of the intrinsic cardiac plexus were isolated for electrophysiological and immunohistochemical analyses. Intracellular voltage recordings from intrinsic cardiac neurons demonstrated no significant changes in passive membrane properties or action potential depolarization compared with age-matched controls and sham-operated animals, but afterhyperpolarization duration was increased in PO animals. Neuronal excitability, as determined by the number of action potentials produced with depolarizing stimuli, was differentially increased in phasic neurons derived from PO animals in response to exogenously applied histamine compared with sham and age-matched controls. Conversely, pituitary adenylate cyclase-activating polypeptide-induced increases in intrinsic cardiac neuron evoked AP frequency were similar between control and PO animals. Immunohistochemical analysis demonstrated a twofold increase in the percentage of neurons immunoreactive for neuronal nitric oxide synthase in PO animals compared with control. The density of mast cells within the intrinsic cardiac plexus from PO animals was also increased twofold compared with preparations from control animals. These results indicate that congestive heart failure associated with chronic pressure overload induces a differential remodeling of intrinsic cardiac neurons and upregulation of neuronal responsiveness to specific neuromodulators. intrinsic cardiac nervous system; histamine; pituitary adenylate cyclase-activating polypeptide; mast cells; nitric oxide synthase; intracellular recording

We investigated the effect of inflammation on slow synaptic transmission in myenteric neurons in the guinea pig ileum. Inflammation was induced by the intraluminal injection of trinitrobenzene sulfonate, and tissues were taken for in vitro investigation 6-7 days later. Brief tetanic stimulation of synaptic inputs (20 Hz, 1 s) induced slow excitatory postsynaptic potentials (EPSPs) in 49% and maintained postsynaptic excitation that lasted from 27 min to 3 h in 13% of neurons from the inflamed ileum. These neurons were classified electrophysiologically as AH neurons; 10 were morphological type II neurons, and one was type I. Such long-term hyperexcitability after a brief stimulus is not encountered in enteric neurons of normal intestine. Electrophysiological properties of neurons with maintained postsynaptic excitation were similar to those of neurons with slow EPSPs. Another form of prolonged excitation, sustained slow postsynaptic excitation (SSPE), induced by 1-Hz, 4-min stimulation, in type II neurons from the inflamed ileum reached its peak earlier but had lower amplitude than that in control. Unlike slow EPSPs and similar to SSPEs, maintained excitation was not inhibited by neurokinin-1 or neurokinin-3 receptor antagonists. Maintained postsynaptic excitation was not influenced by PKC inhibitors, but the PKA inhibitor, H-89, caused further increase in neuronal excitability. In conclusion, maintained excitation, observed only in neurons from the inflamed ileum, may contribute to the dysmotility, pain, and discomfort associated with intestinal inflammation. enteric nervous system; myenteric neurons

Pituitary adenylate cyclase-activating polypeptide (PACAP) increases excitability of guinea pig cardiac neurons, an effect mediated by PACAP-selective [PAC.sub.1] receptors. In dissociated guinea pig cardiac neurons, PACAP causes a positive shift of the voltage dependence of activation of the hyperpolarization-activated nonselective cation current ([I.sub.h]). This observation suggested that an enhancement of [I.sub.h] contributed to the increase in excitability in neurons within whole-mount cardiac ganglia preparations. To evaluate the role of [I.sub.h] in the PACAP-induced increase in excitability, we compared the increase in action potentials generated by 10 nM PACAP in control neurons and in neurons treated with ZD7288 (10 or 100 [micro]M) or CsCl (2 or 2.5 mM), drugs known to inhibit [I.sub.h]. In control cells exposed to PACAP, I-s depolarizing current pulses elicited multiple action potential firing in 79% of the neurons. In ZD7288- or CsCl-containing solutions, the 10 nM PACAP-induced increase in excitability was markedly suppressed, with 7% and 21% of the neurons generating multiple action potentials, respectively. Prior results indicated that PACAP initiates depolarization by activating an inward current, which is separate from its enhancement of [I.sub.h]. Here, we show that a PACAP-induced depolarization was comparable in control neurons and neurons bathed in a CsCl-containing solution, an observation indicating that CsCl did not interfere with activation of the [PAC.sub.1] receptor by PACAP. Additional experiments indicated that pretreatment with the putative M current ([I.sub.M]) inhibitor 1 mM Ba[Cl.sub.2], but not 10 [micro]M XE991, initiated multiple firing in a majority of neurons, with resting potentials maintained at approximately -60 mV. Furthermore, in [Ba.sub.2+]-treated cells, 10 nM PACAP increased the number of action potentials generated. Our results indicate that PACAP enhancement of [I.sub.h], rather than inhibition of [I.sub.M] and other 1 mM [Ba.sub.2+]-sensitive [K.sub.+] currents, is a key ionic mechanism contributing to the peptide-induced increase in excitability for neurons within wholemount cardiac ganglia preparations. hyperpolarization-activated nonselective cation current; neuronal excitability; neuropeptide; parasympathetic neurons

By comparison with murine rodents such as rats, the middle ear structures of many subterranean mammals appear to be enlarged and thus adapted toward low-frequency sound transmission. However, comparison with closely related terrestrial outgroups has not always been undertaken, and apparent specializations in some cases might reflect phylogeny rather than habitat. Examination of the middle ear of the nonsubterranean degu (Octodon degus) under light microscopy revealed a septated middle ear cavity, a circular tympanic membrane lacking a pars flaccida, a malleus with elongated head, synostosed with the incus, a typically bicrurate stapes, and no stapedius muscle. Many of these features are shared with closely related, subterranean octodontoids in the genera Ctenomys (tuco-tucos) and Spalacopus (coruro). Caviomorph rodents in general share a very similar middle ear morphology, regardless of habitat, which suggests that sensitive low-frequency hearing is plesiomorphic for this group, rather than being specifically associated with a subterranean lifestyle. Key words: Cavia, caviomorph, Chinchilla, Ctenomys, degu, middle ear, Octodon, Rattus, rodent, subterranean

Real-time simultaneous detection of changes in pH and levels of histamine over the oxyntic glands of guinea pig stomach have been investigated. An iridium oxide pH microelectrode was used in a potentiometric mode to record the pH decrease associated with acid secretion when the sensor approached the isolated tissue. A borondoped diamond (BDD) microelectrode was used in an amperometric mode to detect histamine when the electrode was placed over the tissue. Both sensors provided stable and reproducible responses that were qualitatively consistent with the signaling mechanism for acid secretion at the stomach. Simultaneous measurements in the presence of pharmacological treatments produced significant variations in the signals obtained by both sensors. As the H2 receptor antagonist cimetidine was perfused to the tissue, histamine levels increased that produced an increase in the signal of the BDD electrode whereas the pH sensor recorded a decrease in acid secretion as expected. Addition of acetylcholine (ACh) stimulated additional acid secretion detected with the pH microelectrode whereas the BDD sensor recorded the histamine levels decreasing significantly. This result shows that the primary influence of ACh is directly on the parietal cell receptors rather then the ECL cell receptors of the oxyntic glands. These results highlight the power of this simultaneous detection technique in the monitoring and diagnosis of physiological significant signaling mechanisms and pathways.

[gamma]-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system and exerts its actions via both ionotropic ([GABA.sub.A]) channels and metabotropic ([GABA.sub.B]) receptors. [GABA.sub.A] channels are ubiquitously expressed in neuronal tissues, and in mature neurons modulate an inward chloride current resulting in neuronal inhibition due to membrane hyperpolarization. In airway smooth muscle (ASM) cells, membrane hyperpolarization favors smooth muscle relaxation. Although [GABA.sub.A] channels and [GABA.sub.B] receptors have been functionally identified on peripheral nerves in the lung, [GABA.sub.A] channels have never been identified on ASM itself. We detected the mRNA encoding of the [GABA.sub.A] [[alpha].sub.4]-, [[alpha].sub.5]-, [[beta].sub.3]-, [[delta], [[gamma].sub.1-3]-[pi]-, and [theta]-subunits in total RNA isolated from native human and guinea pig ASM and from cultured human ASM cells. Selected immunoblots identified the [GABA.sub.A] [[alpha].sub.4]-, [[alpha].sub.5]-, [[beta].sub.3]-, and [[gamma].sub.2]-subunit proteins in native human and guinea pig ASM and cultured human ASM cells. The [GABA.sub.A] [[beta].sub.3]-subunit protein was immunohistochemically localized to ASM in guinea pig tracheal rings. While muscimol, a specific [GABA.sub.A] channel agonist, did not affect the magnitude or the time to peak contractile effect of substance P, it directly concentration dependently relaxed a tachykinin-induced contraction in guinea pig tracheal rings, which was inhibited by the [GABA.sub.A]-selective antagonist gabazine. Muscimol also relaxed a contraction induced by an alternative contractile agonist histamine. These results demonstrate that functional [GABA.sub.A] channels are expressed on ASM and suggest a novel therapeutic target for the relaxation of ASM in diseases such as asthma and chronic obstructive lung disease. RT-PCR; immunoblot; tachykinin; guinea pig; organ bath', histamine

The purpose of this study was to elucidate the mechanisms by which ATP increases guinea pig gallbladder smooth muscle (GBSM) excitability. We evaluated changes in membrane potential and action potential (AP) frequency in GBSM by use of intracellular recording. Application of ATP (100 [micro]M) caused membrane depolarization and a significant increase in AP frequency that were not sensitive to block by tetrodotoxin (0.5 [micro]M). The nonselective P2 antagonist, suramin (100 [micro]M), blocked the excitatory response, resulting in decreased AP frequency in the presence of ATP. The excitatory response to ATP was not altered by pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid (30 [micro]M), a nonselective P2X antagonist. UTP also caused membrane depolarization and increased AP frequency, with a similar dose-response relationship as ATP. RT-PCR demonstrated that the P2[Y.sub.4], but not P2[Y.sub.2], receptor subtype is expressed in guinea pig gallbladder muscularis. ATP induced excitation was blocked by indomethacin (10 [micro]M) and the cyclooxygenase (COX)-1 inhibitor SC-560 (300 nM), but not the COX-2 inhibitor nimesulide (500 nM). These data suggest that ATP stimulates P2[Y.sub.4] receptors within the gallbladder muscularis and, in turn, stimulate prostanoid production via COX-1 leading to increased excitability of GBSM. biliary motility; cyclooxygenase; purinergic; P2Y; prostaglandins

Airway smooth muscle (ASM) from infant guinea pigs has less spontaneous relaxation during stimulation than ASM from adults. Inhibition of cyclooxygenase (COX), which catalyzes the production of prostanoids, increases this relaxation in infant ASM and abolishes age differences, thus suggesting that prostanoids reduce relaxation in infant ASM. In this study, we investigated whether leukotrienes are also involved in reducing spontaneous relaxation; whether the two COX isoforms, COX-1 and COX-2, differentially regulate spontaneous relaxation; and whether prostanoid release is developmentally regulated in guinea pig ASM. In different age groups, we measured relaxation during and after electrical stimulation in tracheal strips as well as prostanoid release from tracheal segments. Relaxation was studied in the absence and in the presence of a lipoxygenase inhibitor, a cysteinyl leukotriene receptor-1 antagonist, a COX-1 inhibitor, or a COX-2 inhibitor. We found that inhibition of lipoxygenase or cysteinyl leukotriene receptor-I antagonism did not increase spontaneous relaxation at any age, thus excluding a role for leukotrienes in this phenomenon. Inhibition of COX-2, but not COX-1, promoted spontaneous relaxation. The basal release of prostanoids was more abundant in tissue from infant animals and decreased significantly with age. Thromboxane [B.sub.2] was the most abundant metabolite released at all ages. Electrical stimulation and epithelium removal did not affect the age difference in prostanoid release. We conclude that increased basal prostanoid release contributes to the reduced spontaneous relaxation in immature guinea pig ASM compared with older animals. By regulating ASM relaxation, prostanoids may play a role in the airway hyperresponsiveness at a young age. airway hyperresponsiveness; bronchospasm removal; cyclooxygenase; lipoxygenase; ontogenesis

Acetylcholine released from parasympathetic excitatory nerves activates contraction in detmsor smooth muscle. Immunohistochemical labeling of guinea pig detrusor with anti-c-Kit and anti-VAChT demonstrated a close structural relationship between interstitial cells of Cajal (ICC) and cholinergic nerves. The ability of guinea pig bladde,' detrusor ICC to respond to the acetylcholine analog, carbachol, was investigated in enzymatically dissociated cells, loaded with the [Ca.sup.2+] indicator fluo 4AM. ICC fired [Ca.sup.2+] transients in response to stimulation by carbachol (1/10 [micro]M). Their pharmacology was consistent with carbachol-induced contractions in strips of detrusor which were inhibited by 4-DAMP (1 [micro]M), an [M.sub.3] receptor antagonist, but not by the [M.sub.2] receptor antagonist methoctramine (1 [micro]M). The source of [Ca.sub.2+] underlying the carbachol transients in isolated 1CC was investigated using agents to interfere with influx or release from intracellular stores. Nifedipine (1 [micro]M) or [Ni.sup.2+] (30-100 [micro]M) to block [Ca.sup.2+] channels or the removal of external [Ca.sup.2+] reduced the amplitude of the carbachol transients. Application of ryanodine (30 [micro]M) or tetracaine (100 [micro]M) abolished the transients. The phospholipase C inhibitor, U-73122 (2.5 [micro]M), significantly reduced the responses. 2-Aminoethoxydiethylborate (30 [micro]M) caused a significant reduction and Xestospongin C (1 [micro]M) was more effective, almost abolishing the responses. Intact in situ preparations of guinea pig bladder loaded with a [Ca.sup.2+] indicator showed distinctively different patterns of spontaneous [Ca.sup.2+] events in smooth muscle ceils and ICC. Both cell types responded to carbachol by an increase in frequency of these events. In conclusion, guinea pig bladder detrusor ICC, both as isolated cells and within whole tissue preparations, respond to cholinergic stimulation by firing [Ca.sup.2+] transients. confocal microscopy; c-Kit

Previous studies have reported on propagation of individual spikes in isolated segments of the pregnant uterus, but there is no information on patterns of spike propagation in the intact organ. There is also no information on propagation of myometrial burst. The aim of this study was to record, at high resolution, patterns of propagation of electrical activities in the pregnant uterus. Sixteen timed-pregnant guinea pigs were euthanized at term, and their uteruses isolated. Fetuses were removed and replaced by an equal amount of Tyrode. A 240-electrode array was positioned at various locations along the organ, all signals were recorded simultaneously, and the electrical propagations were reconstructed. In the intact pregnant uterus at term, spikes propagated with high velocity in longitudinal (6.8 [+ or -] 2.4 cm/s) and slower velocity in circular direction (2.8 [+ or -] 1.0 cm/s; P < 0.01). Direction of propagation and frequency of activity were highly variable but showed similar patterns at the ovary or cervical end and along the anterior, posterior, and antimesometrial borders. Along mesometrium, spike propagation was sparse and fractionated. Migration of burst (0.6 [+ or -] 0.4 cm/s) was significantly much slower than that of individual spikes (P < 0.001). Initial burst activity was located at variable locations along the ovarial end of the antimesometrial border, while the latest excitation occurred at the cervical end (1.2 [+ or -] 0.9 min). In conclusion, high resolution electrical mapping of the intact pregnant uterus reveals fundamental properties in spatial and temporal patterns of spike and burst propagation that determine the contraction of the organ. spikes; myometrial burst migration

Although [[beta].sub.2]-adrenoceptors represent 15-25% of [beta]-adrenoceptors in the guinea pig heart, their functionality is controversial. We assessed the inotropic effects of [[beta].sub.2]-adrenoceptor partial agonists in right papillary muscles. Salbutamol induced a small but significant concentration-dependent negative inotropic effect (NIE, -5% at 60 nM) followed by a moderate positive inotropic effect (+ 36% at 6 [micro]M) due to activation of [[beta].sub.1]-adrenoceptors. In the presence of 4 [micro]M atenolol, the concentration-dependent NIE (- 12% at 6 [micro]M) was biphasic, best described by a double logistic equation with respective [EC.sub.50] values of 3 and ~420 nM, and was insensitive to SR59230A. In muscles from pertussis toxin-treated guinea pigs, the salbutamol-induced positive inotropic effect was sensitive to low concentrations of ICI-118551 in an unusual manner. Experiments in reserpinized animals revealed the importance of the phosphorylation-dephosphorylation processes. PKA inhibition reduced and suppressed the effects obtained at low and high concentrations, respectively, indicating that its activation was a prerequisite to the NIE. The effect occurring at nanomolar concentrations depended upon PKA/phosphatidylinositol 3-kinase/ cytosolic phospholipase [A.sub.2] ([cPLA.sub.2]) activations leading to nitric oxide (NO) release via the arachidonic acid/cyclooxygenase pathway. NO release via PKA-dependent phosphorylation of the receptor was responsible for the inotropic effect observed at submicromolar concentrations, which is negatively controlled by [cPLA.sub.2]. The possibility that these effects are due to an equilibrium between different affinity states of the receptor ([G.sub.s]/[G.sub.i] coupled and [G.sub.i] independent with different signaling pathways) that can be displaced by ICI-118551 is discussed. We conclude that [[beta].sub.2]-adrenoceptors are functional in guinea pig heart and can modulate the inotropic state. salbutamol; [beta]-adrenoceptor antagonists; cardiac contractility; [G.sub.s]/ -[G.sub.i] coupling; active conformations

The role of arachidonic acid (AA) on the development of adipose tissue is still controversial since its metabolites, i.e., prostaglandins, can either stimulate or inhibit preadipocyte differentiation in vitro. In the present study, we evaluated the effects of early postnatal supplementation of AA on body weight and adipose tissue development in guinea pigs. Male newborn guinea pigs were fed for 21 days (day 21) with diets (milk and pellet) supplemented (+ AA) or not (-AA) with 1.2% (total fatty acids) AA. From day 21 to day 105 both groups were fed a chow diet. The 21-days-old +AA pups showed a twofold higher AA accretion in phospholipids associated with a two- to sixfold increase in several prostaglandins, such as 6-keto [PGF.sub.1[alpha]] (the stable hydrolysis product of [PGI.sub.2]), [PGF.sub.2[alpha]], [PGE.sub.2], and [PGD.sub.2] in adipose tissue, compared with the -AA group. No difference in fat pad and body weight, aP2, and leptin gene expression in adipose tissue, fasting plasma glucose, free-fatty acids, and triglyceride concentration was observed between groups at day 21 or day 105. These results show that dietary supplementation of AA during the suckling/weaning period increases prostaglandin levels in adipose tissue but does not influence early fat mass development in the guinea pig. dietary fat; polyunsaturated fatty acids; eicosanoids; metabolic programming; obesity

Our group (Horner KC, Guieu R, Magnan J, Chays A, Cazals Y. Neuropsychopharmacology 26: 135-138, 2002) has earlier described hyperprolactinemia in some patients presenting inner ear dysfunction. However, in that study, it was not possible to determine whether hyperprolactinemia was a cause or an effect of the symptoms. To investigate the effect of hyperprolactinemia on inner ear function, we first developed a model of hyperprolactinemia in estrogen-primed Fischer 344 rats and then performed functional studies on pigmented guinea pigs. Hyperprolactinemia induced, after 2 mo, a hearing loss of ~30-40 dB across all frequencies, as indicated by the compound action potential audiogram. During the 3rd mo, the hearing loss continued to deteriorate. The threshold shifts were more substantial in males than in females. Observations under a dissection microscope revealed bone dysmorphology of the bulla and the cochlea. Light microscopy observations of cryostat sections confirmed bone-related pathology of the bony cochlear bulla and the cochlear wall and revealed morphopathology of the stria vascularis and spiral ligament. Scanning electron microscopy revealed loss of hair cells and stereocilia damage, in particular in the upper three cochlear turns and the two outermost hair cell rows. The data provide the first evidence of otic capsule and hair cell pathology associated with estrogen-induced prolonged hyperprolactinemia and suggest that conditions such as pregnancy, anti-psychotic drug treatment, aging, and/or stress might lead to similar ear dysfunctions. hormones; deafness; prolactin; estrogen; osteoprotegerin

The effects of intracellular [Cl.sup.-] concentration ([[[Cl.sup.-]].sup.i]) on acetylcholine (ACh)-stimulated exocytosis were studied in guinea pig antral mucous cells by video microscopy. ACh activated [Ca.sup.2+]-regulated exocytosis (an initial phase followed by a sustained phase). Bumetanide (20 [micro]M) or a [Cl.sup.-]-free (N[O.sup.-.sub.3]) solution enhanced it; in contrast, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a [Cl.sub.-] channel blocker) decreased it and eliminated the enhancement induced by bumetanide or N[O.sup-.sub.3] solution. ACh and [Ca.sup.2+] dose-response studies demonstrated that N[O.sup-.sub.3] solution does not shift their dose-response curves, and ATP depletion studies by dinitrophenol or anoxia demonstrated that exposure of N[O.sup-.sub.3] solution prior to ATP depletion induced an enhanced initial phase followed by a sustained phase, whereas exposure of N[O.sup-.sub.3] solution after ATP depletion induced only a sustained phase. Intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sup.i]) measurements showed that bumetanide and N[O.sup-.sub.3] solution enhanced the ACh-stimulated [[[Ca.sup.2+]].sup.i] increase. Measurements of [[[Cl.sub.-]].sub.i] revealed that ACh decreases [[[C1.sup.-]].sub.i] and that bumetanide and N[O.sup-.sub.3] solution decreased [[[C1.sup.-]].sub.i] and enhanced the ACh-evoked [[[Cl.sub.-]].sub.i] decrease; in contrast, NPPB increased [[[C1.sub.-]].sub.i] and inhibited the [[[Cl.sub.-]].sub.i] decrease induced by ACh, bumetanide, or N[O.sup-.sub.3] solution. These suggest that [[[Cl.sub.-]].sub.i] modulates [[[Ca.sup.2+]].sub.i] increase and ATP-dependent priming. In conclusion, a decrease in [[[C1.sub.-]].sub.i] accelerates ATP-dependent priming and [[[Ca.sup.2+]].sub.i] increase, which enhance [Ca.sup.2+]-regulated exocytosis in ACh-stimulated antral mucous cells. gastric antrum; mucin exocytosis; acetylcholine; intracellular [Cl.sub.-] concentration

The motility of the gut depends on the chemicals contained in the lumen, but the stimuli that modify motility and their relationship to enteric neural pathways are unclear. This study examined local inhibitory reflexes activated by various chemical stimulants applied to the mucosa to characterize effective physiological stimuli and the pathways they excite. Segments of the jejunum were dissected to allow access to the circular muscle on one-half of the preparation while leaving the mucosa intact on the circumferentially adjacent half. Chemicals were transiently applied to the mucosa, and responses were recorded intracellularly in nearby circular muscle cells. The amino acids e-phenylalanine, L-alanine, or L-tryptophan (all 1 mM) evoked inhibitory junction potentials (IJPs; latency 150-300 ms, amplitude 3-8 mV, each n > 6) that were blocked by TTX and partially blocked by antagonists of P2X receptors and/or a combination of antagonists at 5-H[T.sub.3] and 5-H[T.sub.4] receptors. The putative mediators 5-HT (10 [micro]M), ATP (1 mM), and CCK-8 (1-10 [micro]M) elicited IJPs mediated via 5-H[T.sub.3], P2X, and CCK-B receptors, respectively. Responses were only partially reduced by the effective antagonists. IJPs evoked by electrically stimulating the mucosa were unaffected by antagonists that reduced chemically evoked responses. Both chemically and electrically evoked IJPs were resistant to nicotinic, NKI, N[K.sub.3], [alpha]-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, N-methyl-D-aspartate, or CGRP receptor blockade. We conclude that mucosal stimulation by amino acids activates local neural pathways whose pharmacology depends on the nature of the stimulus. Transmitters involved at some synapses in these pathways remain to be identified. 5-HT3 receptors; P2X receptors; cholecystokinin receptors; enteric reflexes doi:10.1152/ajpgi.00580.2006

Mechanisms underlying nutrient-induced segmentation within the gut are not well understood. We have shown that decanoic acid and some amino acids induce neurally dependent segmentation in guinea pig small intestine in vitro. This study examined the neural mechanisms underlying segmentation in the circular muscle and whether the timing of segmentation contractions also depends on slow waves. Decanoic acid (1 mM) was infused into the lumen of guinea pig duodenum and jejunum. Video imaging was used to monitor intestinal diameter as a function of both longitudinal position and time. Circular muscle electrical activity was recorded by using suction electrodes. Recordings from sites of segmenting contractions showed they are always associated with excitatory junction potentials leading to action potentials. Recordings from sites oral and anal to segmenting contractions revealed inhibitory junction potentials that were time locked to those contractions. Slow waves were never observed underlying segmenting contractions. In paralyzed preparations, intracellular recording revealed that slow-wave frequency was highly consistent at 19.5 (SD 1.4) cycles per minute (c/min) in duodenum and 16.6 (SD 1.1) c/min in jejunum. By contrast, the frequencies of segmenting contractions varied widely (duodenum: 3.6-28.8 c/min, median 10.8 c/min; jejunum: 3.0-27.0 c/min, median 7.8 c/min) and sometimes exceeded slow-wave frequencies for that region. Thus nutrient-induced segmentation contractions in guinea pig small intestine do not depend on slow-wave activity. Rather they result from a neural circuit producing rhythmic localized activity in excitatory motor neurons, while simultaneously activating surrounding inhibitory motor neurons. enteric nervous system; intestinal motility patterns; slow waves

Appropriate partitioning of nutrients between the mother and conceptus is a major determinant of pregnancy success, with placental transfer playing a key role. Insulin-like growth factors (IGFs) increase in the maternal circulation during early pregnancy and are predictive of fetal and placental growth. We have previously shown in the guinea pig that increasing maternal IGF abundance in early to midpregnancy enhances fetal growth and viability near term. We now show that this treatment promotes placental transport to the fetus, fetal substrate utilization, and nutrient partitioning near term. Pregnant guinea pigs were infused with IGF-I, IGF-II (both 1 mg*[kg.sup.-1]*[day.sup.-1]) or vehicle subcutaneously from days 20-38 of pregnancy (term = 69 days). Tissue uptake and placental transfer of the nonmetabolizable radio analogs [[sup.3]H]methyl-D-glucose (MG) and [[sup.14]C]aminoisobutyric acid (AIB) in vivo was measured on day 62. Early pregnancy exposure to elevated maternal IGF-I increased placental MG uptake by >70% (P = 0.004), whereas each IGF increased fetal plasma MG concentrations by 40-50% (P < 0.012). Both IGFs increased fetal tissue MG uptake (P < 0.048), whereas IGF-I also increased AIB uptake by visceral organs (P = 0.046). In the mother, earlier exposure to either IGF increased AIB uptake by visceral organs (P < 0.014), whereas IGF-I also enhanced uptake of AIB by muscle (P = 0.044) and MG uptake by visceral organs (P = 0.016) and muscle (P = 0.046). In conclusion, exogenous maternal IGFs in early pregnancy sustainedly increase maternal substrate utilization, placental transport of MG to the fetus, and fetal utilization of substrates near term. This was consistent with the previously observed increase in fetal growth and survival following IGF treatment. insulin-like growth factor; fetal growth; glucose transport; system A amino acid transport

This study examined whether mucosal stimulation activates long secretomotor neural reflexes and, if so, how they are organized. The submucosa of in vitro full thickness guinea pig ileal preparations was exposed in the distal portion and intracellular recordings were obtained from electrophysiologically identified secretomotor neurons. Axons in the intact mucosa of the oral segment were stimulated by a large bipolar stimulating electrode. In control preparations, a single stimulus pulse evoked a fast excitatory postsynaptic potential (EPSP) in 86% of neurons located 0.7-1.0 cm anal to the stimulus site. A stimulus train evoked multiple fast EPSPs, but slow EPSPs were not observed. To examine whether mucosal stimulation specifically activated mucosal sensory nerve terminals, the mucosa/submucosa was severed from the underlying layers and repositioned. In these preparations, fast EPSPs could not be elicited in 89% of cells. Superfusion with phorbol dibutyrate enhanced excitability of sensory neurons and pressure-pulse application of serotonin to the mucosa increased the fast EPSPs evoked by mucosal stimulation, providing further evidence that sensory neurons were involved. To determine whether these reflexes projected through the myenteric plexus, this plexus was surgically lesioned between the stimulus site and the impaled neuron. No fast EPSPs were recorded in these preparations following mucosal stimulation whereas lesioning the submucosal plexus had no effect. These results demonstrate that mucosal stimulation triggers a long myenteric pathway that activates submucosal secretomotor neurons. This pathway projects in parallel with motor and vasodilator reflexes, and this common pathway may enable coordination of intestinal secretion, blood flow, and motility. secretomotor reflexes; submucosa; secretion

Actions of adenosine 5'-monophosphate (AMP) on electrical and synaptic behavior of submucosal neurons in guinea pig small intestine were studied with 'sharp' intracellular microelectrodes. Application of AMP (0.3-100 [micro]M) evoked slowly activating depolarizing responses associated with increased excitability in 80.5% of the neurons. The responses were concentration dependent with an ECso of 3.5 [+ or -] 0.5 [micro]M. They were abolished by the adenosine [A.sub.2A] receptor antagonist ZM-241385 but not by pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid, trinitrophenyl-ATP, 8-cyclopentyl-1,3-dimethylxanthine, suramin, or MRS-12201220. The AMP-evoked responses were insensitive to AA-COCF3 or ryanodine. They were reduced significantly by 1) U-73122, which is a phospholipase C inhibitor; 2) cyclopiazonic acid, which blocks the [Ca.sup.2+] pump in intraneuronal membranes; and 3) 2-aminoethoxy-diphenylborane, which is an inositol (1,4,5)-trisphosphate receptor antagonist. Inhibitors of PKC or calmodulin-dependent protein kinase also suppressed the AMP-evoked excitatory responses. Exposure to AMP suppressed fast nicotinic ionotropic postsynaptic potentials, slow metabotropic excitatory postsynaptic potentials, and slow noradrenergic inhibitory postsynaptic potentials in the submucosal plexus. Inhibition of each form of synaptic transmission reflected action at presynaptic inhibitory adenosine [A.sub.1] receptors. Slow excitatory postsynaptic potentials, which were mediated by the release of ATP and stimulation of P2[Y.sub.1] purinergic receptors in the submucosal plexus, were not suppressed by AMP. The results suggest an excitatory action of AMP at adenosine [A.sub.2A] receptors on neuronal cell bodies and presynaptic inhibitory actions mediated by adenosine [A.sub.1] receptors for most forms of neurotransmission in the submucosal plexus, with the exception of slow excitatory purinergic transmission mediated by the P2[Y.sub.1] receptor subtype. gastrointestinal tract; neurogastroenterology; enteric nervous system; synaptic transmission; purinergic receptors

The role of enteric glia in gastrointestinal physiology remains largely unexplored. We examined the actions of the gliotoxin fluorocitrate (FC) on intestinal motility, secretion, and inflammation after assessing its efficacy and specificity in vitro. FC (100 [micro]M) caused a significant decrease in the phosphorylation of the glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diaz-4-yl) amino]-2-deoxyglucose in enteric glial cultures and a reduction in glial uptake of the fluorescent dipeptide Ala-Lys-7-amino-4-methylcoumarin-3-acetic acid in both the ileum and colon. Dipeptide uptake by resident murine macrophages or guinea pig myenteric neurons was unaffected by FC. Incubation of isolated guinea pig ileal segments with FC caused a specific and significant increase in glial expression of the phosphorylated form of ERK-1/2. Disruption of enteric glial function with FC in mice reduced small intestinal motility in vitro, including a significant decrease in basal tone and the amplitude of contractility in response to electrical field stimulation. Mice treated with 10 or 20 [micro]mol/kg FC twice daily for 7 days demonstrated a concentration-dependent decrease in small intestinal transit. In contrast, no changes in colonic transit or ion transport in vitro were observed. There were no changes in glial or neuronal morphology, any signs of inflammation in the FC-treated mice, or any change in the number of myenteric nitric oxide synthase-expressing neurons. We conclude that FC treatment causes enteric glial dysfunction, without causing intestinal inflammation. Our data suggest that enteric glia are involved in the modulation of enteric neural circuits underlying the regulation of intestinal motility. enteric glia; myenteric plexus; colonic ion transport

Activation of P2X receptors by a [Ca.sup.2+]--and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-dependent release of ATP was measured using patch-clamp recordings from dissociated guinea pig stellate neurons. Asynchronous transient inward currents (ASTICs) were activated by depolarization or treatment with the [Ca.sup.2+] ionophore ionomycin (1.5 and 3 [micro]M). During superfusion with a HEPES-buffered salt solution containing 2.5 mM [Ca.sup.2+], depolarizing voltage steps (-60 to 0 mV, 500 ms) evoked ASTICs on the decaying phase of a larger, transient inward current. Equimolar substitution of [Ba.sup.2+] for [Ca.sup.2+] augmented the postdepolarization frequency of ASTICs, while eliminating the larger transient current. Perfusion with an ionomycin-containing solution elicited a sustained activation of ASTICs, allowing quantitative analysis over a range of holding potentials. Under these conditions, increasing extracellular [[Ca.sup.2+]] to 5 mM increased ASTIC frequency, whereas no events were observed following replacement of [Ca.sup.2+] with [Mg.sup.2+], demonstrating a [Ca.sup.2+] requirement. ASTICs were [Na.sup.+] dependent, inwardly rectifying, and reversed near 0 mV. Treatment with the nonselective purinergic receptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (10 [micro]M) blocked all events under both conditions, whereas the ganglionic nicotinic antagonist hexamethonium (100 [micro]M and 1 mM) had no effect. PPADS also blocked the macroscopic inward current evoked by exogenously applied ATP (300 [micro]M). The presence of botulinum neurotoxin E (BoNT/E) in the whole-cell recording electrode significantly attenuated the ionomycin-induced ASTIC activity, whereas phorbol ester treatment potentiated this activity. These results suggest that ASTICs are mediated by vesicular release of ATP and activation of P2X receptors. sympathetic; purinergic; neurotransmission; phorbol ester; botulinum toxin

We are developing two types of vestibular prosthetics that electrically stimulate afferent neurons. One type replaces absent sensory function by providing stimulation that modulates above and below a baseline established with the head stationary. The other type provides constant stimulation and is turned on only when necessary, for example, to override unnatural variations like those experienced by patients suffering from Meniere's syndrome; this prosthesis does not provide motion information. Both prostheses require neural plasticity, which we investigated by providing chronic constant-rate stimulation to semicircular canal neurons in three guinea pigs. The stimulation was alternately switched on or off for eight consecutive weeks before being switched daily. A brisk horizontal nystagmus was measured when the stimulation was first turned on and then dissipated over the course of a day. The nystagmus demonstrated an after-effect in the opposite direction when the stimulation was turned off. The nystagmus that we measured after just a few (2 to 5) off-to-on transitions returned to baseline more rapidly than when first turned on. In fact, after many such off-to-on or on-to-off transitions, little nystagmus was evoked by turning the stimulation on or off. These findings show that the brain acclimates to constant-rate stimulation. Index Terms--Adaptation, guinea pig, neural prosthesis, vestibular implant, vestibulo-ocular reflex, VOR.

The nerve agents soman, sarin, VX, and tabun are deadly organophosphorus (OP) compounds chemically related to OP insecticides. Most of their acute toxicity results from the irreversible inhibition of acetylcholinesterase (ACHE), the enzyme that inactivates the neurotransmitter acetylcholine. The limitations of available therapies against OP poisoning are well recognized, and more effective antidotes are needed. Here, we demonstrate that galantamine, a reversible and centrally acting AChE inhibitor approved for treatment of mild to moderate Alzheimer's disease, protects guinea pigs from the acute toxicity of lethal doses of the nerve agents soman and sarin, and of paraoxon, the active metabolite of the insecticide parathion. In combination with atropine, a single dose of galantamine administered before or soon after acute exposure to lethal doses of soman, sarin, or paraoxon effectively and safely counteracted their toxicity. Doses of galantamine needed to protect guinea pigs fully against the lethality of OPs were well tolerated. In preventing the lethality of nerve agents, galantamine was far more effective than pyridostigmine, a peripherally acting AChE inhibitor, and it was less toxic than huperzine, a centrally acting AChE inhibitor. Thus, a galantamine-based therapy emerges as an effective and safe countermeasure against OP poisoning. galantamine | guinea pig | pyridostigmine | soman | sarin

The transient outward current ([I.sub.to]) is a major repolarizing current in the heart. Marked reduction of Ito density occurs in heart failure and is accompanied by significant action potential duration (APD) prolongation. To understand the species-dependent role of [I.sub.to] in regulating the ventricular action potential morphology and duration, we introduced simulated [I.sub.to] conductance in guinea pig and canine endocardial ventricular myocytes using the dynamic clamp technique and perforated patch-clamp recordings. The effects of simulated [I.sub.to] in both types of cells were complex and biphasic, separated by a clear density threshold of ~40 pA/pF. Below this threshold, simulated [I.sub.to] resulted in a distinct phase 1 notch and had little effect on or moderately prolonged the APD. [I.sub.to] above the threshold resulted in all-or-none repolarization and precipitously reduced the APD. Qualitatively, these results agreed with our previous studies in canine ventricular cells using whole cell recordings. We conclude that 1) contrary to previous gene transfer studies involving the Kv4.3 current, the response of guinea pig ventricular myocytes to a fully inactivating [I.sub.to] is similar to that of canine ventricular cells and 2) in animals such as dogs that have a broad cardiac action potential, [I.sub.to] does not play a major role in setting the APD. dynamic clamp; transient outward current; ventricular myocytes doi:10.1152/ajpheart.00084.2006

Because the onset of fever induced by intravenously (iv) injected bacterial endotoxic lipopolysaccharides (LPS) precedes the appearance in the bloodstream of pyrogenic cytokines, the presumptive peripheral triggers of the febrile response, we have postulated previously that, in their stead, PG[E.sub.2] could be the peripheral fever trigger because it appears in blood coincidentally with the initial body core temperature ([T.sub.c]) rise. To test this hypothesis, we injected Salmonella enteritidis LPS (2 [micro]g/kg body wt iv) into conscious guinea pigs and measured their plasma levels of LPS, PG[E.sub.2], TNF-[alpha], IL-1[beta], and IL-6 before and 15, 30, 60, 90, and 120 min after LPS administration; [T.sub.c]. was monitored continuously. The animals were untreated or Kupffer cell (KC) depleted; the essential involvement of KCs in LPS fever was shown previously. LPS very promptly (< 10 min) induced a rise of T,. that was temporally correlated with the elevation of plasma PG[E.sub.2]. KC depletion prevented the [T.sub.c] and plasma PG[E.sub.2] rises and slowed the clearance of LPS from the blood. TNF-[alpha] was not detectable in plasma until 30 min and in IL-1[beta] and IL-6 until 60 min after LPS injection. KC depletion did not alter the times of appearance or magnitudes of rises of these cytokines, except TNF-[alpha], the maximal level of which was increased approximately twofold in the KC-depleted animals. In a follow-up experiment, PG[E.sub.2] antiserum administered iv 10 min before LPS significantly attenuated the febrile response to LPS. Together, these results support the view that, in guinea pigs, PG[E.sub.2] rather than pyrogenic cytokines is generated by KCs in immediate response to iv LPS and triggers the febrile response. fever; tumor necrosis factor-[alpha]; interleukin-l[beta]; interleukin-6; liver; complement

The aim was to determine the role CGRP and/or tachykinins released from sensory neural mechanisms in enteric neural vasodilator pathways. These pathways project through the myenteric plexus to submucosal vasodilator neurons. Submucosal arterioles were exposed in the distal portion of an in vitro combined submucosal-myenteric guinea pig ileal preparation, and dilation was monitored with videomicroscopy. Vasodilator neural reflexes were activated by gently stroking the mucosa with a fine brush or by distending a balloon placed beneath the flat-sheet preparation in the proximal portion. Dilations evoked by mucosal stroking were inhibited 64% by the CGRP 8-37 and 37% by N[K.sub.3] (SR 142801) antagonists. When the two antagonists were combined with hexamethonium, only a small vasodilation persisted. Balloon distension-evoked vasodilations were inhibited by N[K.sub.3] antagonists (66%) but were not altered by CGRP 8-37. In preparations in which myenteric descending interneurons were directly activated by electrical stimulation, combined application of CGRP 8-37 and the NK antagonists had no effect. Stimulation of capsaicin sensitive nerves in the myenteric plexus did not activate these vasodilator reflexes. These findings suggest that mucosal-activated reflexes result from the release of CGRP and tachykinins from enteric sensory neurons. Distension-evoked responses were significantly blocked by N[K.sub.3] antagonists, suggesting that stretch activation of myenteric sensory neurons release tachykinins that activate N[K.sub.3] receptors on myenteric vasodilator pathways. intrinsic primary afferent neuron; vasodilation; submucosal plexus; submucosal arterioles; tachykinins; calcitonin gene-related peptide

In guinea pigs, dose-dependent febrile responses were induced by injection of a high (100 [micro]g/kg) or a low (10 [micro]g/kg) dose of bacterial lipopolysaccharide (LPS) into artificial subcutaneously implanted Teflon chambers. Both LPS doses further induced a pronounced formation of prostaglandin [E.sub.2] (PG[E.sub.2]) at the site of localized subcutaneous inflammation. Administration of diclofenac, a nonselective cyclooxygenase (COX) inhibitor, at different doses (5, 50, 500, or 5,000 [micro]g/kg) attenuated or abrogated LPS-induced fever and inhibited LPS-induced local PG[E.sub.2] formation (5 or 500 [micro]g/kg diclofenac). Even the lowest dose of diclofenac (5 [micro]g/kg) attenuated fever in response to 10 [micro]g/kg LPS, but only when administered directly into the subcutaneous chamber, and not into the site contralateral to the chamber. This observation indicated that a localized formation of PG[E.sub.2] at the site of inflammation mediated a portion of the febrile response, which was induced by injection of 10 [micro]g/kg LPS into the subcutaneous chamber. Further support for this hypothesis derived from the observation that we failed to detect elevated amounts of COX-2 mRNA in the brain of guinea pigs injected subcutaneously with 10 [micro]g/kg LPS, whereas subcutaneous injections of 100 [micro]g/kg LPS, as well as systemic injections of LPS (intra-arterial or intraperitoneal routes), readily caused expression of the COX-2 gene in the guinea pig brain, as demonstrated by in situ hybridization. Therefore, fever in response to subcutaneous injection of 10 [micro]g/kg LPS may, in part, have been evoked by a neural, rather than a humoral, pathway from the local site of inflammation to the brain. lipopolysaccharide; febrile response; prostaglandin [E.sub.2]; cyclooxygenase-2; immune system-to-brain communication

Respiratory tract lesions induced by the chemical warfare agent sulfur mustard (SM) are characterized by epithelial damages associated with inflammatory cell infiltration. Here we evaluated the imbalance between gelatinase and tissue inhibitors of metalloproteinases (TIMPs), and we tested pretreatment with the protease inhibitor doxycycline. Guinea pigs were intoxicated intratracheally with SM and evaluated 24 h after exposure. Matrix metalloproteinase (MMP) gelatinase activity of bronchial lavage (BL) fluid from SM-exposed guinea pigs was high compared with controls, as shown by both zymography and biotinylated substrate degradation, whereas TIMP-1 and -2 levels by immunoblotting were similar. Extensive areas of lysis were evidenced by in situ zymography, indicating imbalance between gelatinases and inhibitors towards net proteolytic activity. Doxycycline pretreatment resulted in 1) decreased gelatinase activity (zymography, free gelatinase activity assay, and in situ zymography); 2) decreased inflammation (BL fluid cellularity and protein level); and 3) dramatic decrease in histological epithelial lesions. Our results suggest inadequate levels of TIMP to counteract increased gelatinase activity and further support a role for MMP gelatinases in SM-induced respiratory lesions. They also suggest that doxycycline may hold promise as a therapeutic tool. vesicant agents; blistering agents; proteases; doxycycline; protease inhibitors

Ischemic preconditioning (IPC) induces distinctive changes in mitochondrial bioenergetics during warm (37[degrees]C) ischemia and improves function and tissue viability on reperfusion. We examined whether IPC before 2 h of hypothermic (27[degrees]C) ischemia affords additive cardioprotection and improves mitochondrial redox balance assessed by mitochondrial NADH and ftavin adenine dinucleotide (FAD) autofluorescence in intact hearts. A mediating role of ATP-sensitive [K.sup.+] ([K.sub.ATP]) channel opening was investigated. NADH and FAD fluorescence was measured in the left ventricular wall of guinea pig isolated hearts assigned to five groups of eight animals each: hypothermia alone, hypothermia with ischemia, IPC with cold ischemia, 5-hydroxydecanoic acid (5-HD) alone, and 5-HD with IPC and cold ischemia. IPC consisted of two 5-min periods of warm global ischemia spaced 5 min apart and 15 min of reperfusion before 2 h of ischemia at 27[degrees]C and 2 h of warm reperfusion. The [K.sub.ATP] channel inhibitor 5-HD was perfused from 5 min before until 5 min after IPC. IPC before 2 h of ischemia at 27[degrees]C led to better recovery of function and less tissue damage on reperfusion than did 27[degrees]C ischemia alone. These improvements were preceded by attenuated increases in NADH and decreases in FAD during cold ischemia and the reverse changes during warm reperfusion. 5-HD blocked each of these changes induced by IPC. This study indicates that IPC induces additive cardioprotection with mild hypothermic ischemia by improving mitochondrial bioenergetics during and after ischemia. Because effects of IPC on subsequent changes in NADH and FAD were inhibited by 5-HD, this suggests that mitochondrial [K.sub.ATP] channel opening plays a substantial role in improving mitochondrial bioenergetics throughout mild hypothermic ischemia and reperfusion. nicotinamide adenine dinucleotide; flavin adenine dinucleotide; ATP-sensitive potassium channels; 5-hydroxydecanoic acid

Ethanol strongly augments secretin-stimulated, but not acetylcholine (ACh)-stimulated, fluid secretion from pancreatic duct cells. To understand its mechanism of action, we examined the effect of short-chain n-alcohols on fluid secretion and intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+].sub.i]) in guinea pig pancreatic ducts. Fluid secretion was measured by monitoring the luminal volume of isolated interlobular ducts. [[Ca.sup.2+].sub.i] was estimated using fura-2 microfluorometry. Methanol and ethanol at 0.3-10 mM concentrations significantly augmented fluid secretion and induced a transient elevation of [[Ca.sup.2+].sub.i] in secretin- or dibulyryl adenosine 3',5'-cyclic monophosphate (DBcAMP)-stimulated ducts. However, they failed to affect fluid secretion and [[Ca.sup.2+].sub.i] in unstimulated and ACh-stimulated ducts. In contrast, propanol and butanol at 0.3-10 mM concentrations significantly reduced fluid secretion and decreased [[Ca.sup.2+].sub.i] in unstimulated ducts and in ducts stimulated with secretin, DBcAMP, or ACh. Both stimulatory and inhibitory effects of n-alcohols completely disappeared after their removal from the perfusate. Propanol and butanol inhibited the plateau phase, but not the initial peak, of [[Ca.sup.2+].sub.i], response to ACh as well as the [[Ca.sup.2+].sub.i] elevation induced by thapsigargin, suggesting that they inhibit [Ca.sup.2+] influx. Removal of extracellular [Ca.sup.2+] reduced [[Ca.sup.2+].sub.i] in duct cells and completely abolished secretin-stimulated fluid secretion. In conclusion, there is a distinct cutoff point between ethanol (C2) and propanol (C3) in their effects on fluid secretion and [[Ca.sup.2+].sub.i] in duct cells. Short-chain n-alcohols appear to affect pancreatic ductal fluid secretion by activating or inhibiting the plasma membrane [Ca.sup.2+] channel. intracellular calcium; acetylcholine

Stimulation of [beta]-adrenoceptors contributes to the relaxation of urinary bladder smooth muscle (UBSM) through activation of large-conductance [Ca.sup.2+]-activated [K.sup.+] (BK) channels. We examined the mechanisms by which [beta]-adrenoceptor stimulation leads to an elevation of the activity of BK channels in UBSM. Depolarization from -70 to +10 mV evokes an inward L-type dihydropyridine-sensitive voltage-dependent [Ca.sup.2+] channel (VDCC) current, followed by outward steady-state and transient BK current. In the presence of ryanodine, which blocks the transient BK currents, isoproterenol, a nonselective [beta]-adrenoceptor agonist, increased the VDCC current by ~25% and the steady-state BK current by ~30%. In the presence of the BK channel inhibitor iberiotoxin, isoproterenol did not cause activation of the remaining steady-state [K.sup.+] current component. Decreasing [Ca.sup.2+] influx through VDCC by nifedipine or depolarization to +80 mV suppressed the isoproterenol-induced activation of the steady-state BK current. Unlike forskolin, isoproterenol did not change significantly the open probability of single BK channels in the absence of [Ca.sup.2+] sparks and with VDCC inhibited by nifedipine. Isoproterenol elevated [Ca.sup.2+] spark (local intracellular [Ca.sup.2+] release through ryanodine receptors of the sarcoplasmic reticulum) frequency and associated transient BK currents by ~1.4-fold. The data support the concept that in UBSM [beta]-adrenoceptor stimulation activates BK channels by elevating [Ca.sup.2+] influx through VDCC and by increasing [Ca.sup.2+] sparks, but not through a [Ca.sup.2+]-independent mechanism. This study reveals key regulatory molecular and cellular mechanisms of [beta]-adrenergic regulation of BK channels in UBSM that could provide new targets liar drugs in the treatment of bladder dysfunction. [Ca.sup.2+] sparks; voltage-dependent [Ca.sup.2+] channel; ryanodine receptor

The cardiac [Na.sup.+/[Ca.sup.2+] exchanger (NCX) contributes to cellular injury during hypoxia, as its altered function is largely responsible for a rise in cytosolic [Ca.sup.2+] concentration ([[Ca.sup.2+].sub.i]). In addition, the NCX in guinea pig ventricular myocytes undergoes profound inhibition during hypoxia and rapid reactivation during reoxygenation. The mechanisms underlying these changes in NCX activity are likely complex due to the participation of multiple inhibitory factors including altered cytosolic [Na.sup.+] concentration, pH, and ATP. Our main hypothesis is that oxidative stress is an essential trigger for rapid NCX reactivation in guinea pig ventricular myocytes and is thus a critical factor in determining the timing and magnitude of [Ca.sup.2+] overload. This hypothesis was evaluated in cardiac myocytes using fluorescent indicators to measure [[Ca.sup.2+].sub.i] and oxidative stress. An NCX antisense oligonucleotide was used to decrease NCX protein expression in some experiments. Our results indicate that NCX activity is profoundly inhibited in hypoxic guinea pig ventricular myocytes but is reactivated within 1-2 min of reoxygenation at a time of rising oxidative stress. We also found that several interventions to decrease oxidative stress including antioxidants and diazoxide prevented NCX reactivation and [Ca.sup.2+] overload during reoxygenation. Furthermore, application of exogenous [H.sub.2][O.sub.2] was sufficient by itself to reactivate the NCX during sustained hypoxia and could reverse the suppression of reoxygenation-mediated NCX reactivation by diazoxide. These data suggest that elevated oxidative stress in reoxygenated guinea pig ventricular myocytes is required for rapid NCX reactivation, and thus reactivation should he viewed as an active process rather than being due to the simple decline of NCX inhibition. sodium-calcium exchanger; antioxidants; diazoxide; heart; hypoxia; ischemia

In vitro antigen challenge has multiple effects on the excitability of guinea pig bronchial parasympathetic ganglion neurons, including depolarization, causing phasic neurons to fire with a repetitive action potential pattern and potentiating synaptic transmission. In the present study, guinea pigs were passively sensitized to the antigen ovalbumin. After sensitization, the bronchi were prepared for in vitro electrophysiological intracellular recording of parasympathetic ganglia neurons to investigate the contribution of cyclooxygenase activation and prostanoids on parasympathetic nerve activity. Cyclooxygenase inhibition with either indomethacin or piroxicam before in vitro antigen challenge blocked the change in accommodation. These cyclooxygenase inhibitors also blocked the release of prostaglandin [D.sub.2] (PG[D.sub.2]) from bronchial tissue during antigen challenge. We also determined that PG[E.sub.2] and PG[D.sub.2] decreased the duration of the action potential after hyperpolarization, whereas PG[F.sub.2.sub.[alpha]] potentiated synaptic transmission. Thus prostaglandins released during antigen challenge have multiple effects on the excitability of guinea pig bronchial parasympathetic ganglia neurons, which may consequently affect the output from these neurons and thereby alter parasympathetic tone in the lower airways. asthma; bronchoconstriction; synaptic transmission; ganglia; guinea pig

Submucosal cholinergic and noncholinergic neurons in intestines have been shown to be involved in regulating epithelial transport functions, particularly stimulating [Cl.sup.-] secretion. This study investigates the role of submucosal cholinergic neurons in regulating electrogenic [Na.sup.+] absorption in distal colon. Amiloride-sensitive short-circuit current ([I.sub.sc]) and [sup.22][Na.sup.+] flux were measured in mucosal and mucosal-submucosal preparations mounted in Ussing chambers. In the mucosal preparation, carbachol (CCh) added to the serosal side inhibited amiloride-sensitive [I.sub.sc] and amiloride-sensitive [sup.22][Na.sup.+] absorption. The inhibitory effect of CCh was observed at ~0.1 [micro]M, and maximum inhibition of ~70% was attained at ~30 [micro]M (I[C.sub.50] = ~1 [micro]M). CCh-induced inhibition of amiloride-sensitive [I.sub.sc] was almost totally abolished by 10 [micro]M atropine. Treatment of the tissue with ionomycin markedly reduced amiloride-sensitive [I.sub.sc], but a subsequent addition of CCh further decreased it. Also, CCh still had an inhibitory effect, although significantly attenuated, after the tissue had been incubated with a low-[Ca.sup.2+] solution containing ionomycin and BAPTA-AM. Applying electrical field stimulation to submucosal neurons in the mucosal-submucosal preparation resulted in inhibition of amiloride-sensitive [I.sub.sc], ~33% of this inhibition being atropine sensitive. Physostigmine inhibited amiloride-sensitive [I.sub.sc], this effect being abolished by atropine. In conclusion, submucosal cholinergic and noncholinergic neurons were involved in inhibiting electrogenic [Na.sup.+] absorption in colon. This inhibition by cholinergic neurons was mediated by muscarinic receptor activation. enteric nerve; intracellular [Ca.sup.2+]; acetylcholine; intestinal secretion; epithelial [Na.sup.+] channel

The role of the longitudinal muscle (LM) layer during the peristaltic reflex in the small and large intestine is unclear. In this study, we have made double and quadruple simultaneous intracellular recordings from LM and circular muscle (CM) cells of guinea pig distal colon to correlate the electrical activities in the two different muscle layers during circumferential stretch. Simultaneous recordings from LM and CM cells ( circular muscle; inhibitory junction potential; excitatory junction potential; myenteric neuron; peristaltic reflex; peristalsis

Maternal nutrient restriction and impaired fetal growth are associated with postnatal insulin resistance, hyperinsulinemia, and glucose intolerance in humans but not consistently in other species, such as the rat or sheep. We therefore determined the effect of mild (85% ad libitum intake/kg body wt) or moderate (70% ad libitum intake/kg body wt) maternal feed restriction throughout pregnancy on glucose and insulin responses to an intravenous glucose tolerance test (IVGTT) in the young adult guinea pig. Maternal feed restriction reduced birth weight (mild and moderate: both P < 0.02) in male offspring. Moderate restriction increased plasma glucose area under the curve (P < 0.04) and decreased the glucose tolerance index ([K.sub.G]) (P < 0.02) during the IVGTT in male offspring compared with those of mildly restricted but not of ad libitum-fed mothers. Moderate restriction increased fasting plasma insulin (P < 0.04, adjusted for litter size) and the insulin response to IVGTT (P < 0.001), and both moderate and mild restriction increased the insulin-to-glucose ratio during the IVGTT (P < 0.003 and P < 0.02) in male offspring. When offspring were classed into tertiles according to birth weight, glucose tolerance was not altered, but fasting insulin concentrations were increased in low compared with medium birth weight males (P < 0.03). The insulin-to-glucose ratio throughout the IVGTT was increased in low compared with medium (P < 0.01) or high (P < 0.05) birth weight males. Thus maternal feed restriction in the guinea pig restricts fetal growth and causes hyperinsulinemia in young adult male offspring, suggestive of insulin resistance. These findings suggest that mild to moderate prenatal perturbation programs postnatal glucose homeostasis adversely in the guinea pig, as in the human. birth weight; intravenous glucose tolerance test; plasma insulin

The interlobular duct cells of the guinea-pig pancreas secrete HC[O.sub.3.sup.-] across their luminal membrane into a HC[O.sub.3.sup.-]-rich (125 mM) luminal fluid against a sixfold concentration gradient. Since HC[O.sub.3.sup.-] transport cannot be achieved by luminal Cl-/HC[O.sub.3.sup.-] exchange under these conditions, we have investigated the possibility that it is mediated by an anion conductance. To determine whether the electrochemical potential gradient across the luminal membrane would favor HC[O.sub.3.sup.-] efflux, we have measured the intracellular potential ([V.sub.m]) in microperfused, interlobular duct segments under various physiological conditions. When the lumen was perfused with a 124 mM [Cl.sup.-]-25 mM HC[O.sub.3.sup.-] solution, a condition similar to the basal state, the resting potential was approximately -60 mV. Stimulation with dbcAMP or secretin caused a transient hyperpolarization (~5 mV) due to activation of electrogenic [Na.sup.+]- HC[O.sub.3.sup.-] cotransport at the basolateral membrane. This was followed by depolarization to a steady-state value of approximately -50 mV as a result of anion efflux across the luminal membrane. Raising the luminal HC[O.sub.3.sup.-] concentration to 125 mM caused a hyperpolarization (~10 mV) in both stimulated and unstimulated ducts. These results can be explained by a model in which the depolarizing effect of [Cl.sup.-] efflux across the luminal membrane is minimized by the depletion of intracellular [Cl.sup.-] and offset by the hyperpolarizing effects of [Na.sup.+]- HC[O.sub.3.sup.-] cotransport at the basolateral membrane. The net effect is a luminally directed electrochemical potential gradient for HC[O.sub.3.sup.-] that is sustained during maximal stimulation. Our calculations indicate that the electrodiffusive efflux of HC[O.sub.3.sup.-] to the lumen via CFTR, driven by this gradient, would be sufficient to fully account for the observed secretory flux of HC[O.sub.3.sup.-]. KEY WORDS: pancreatic ducts * membrane potentials * bicarbonate ion * cystic fibrosis transmembrane conductance regulator * sodium-bicarbonate cotransporter

We found that isolated guinea pig spinal cord white matter is resistant to acute oxygen-glucose deprivation. Sixty minutes of oxygen-glucose deprivation resulted in a 60% reduction of compound action potential (CAP) conductance, and there was a near complete recovery after 60 min reperfusion. Corresponding horseradish peroxidase-exclusion assay showed little axonal membrane damage. To further deprive the axons of metabolic substrate, we added 2 mM sodium cyanide or 2 mM sodium azide, both mitochondrial suppressors, to the ischemic medium, which completely abolished CAP and resulted in a 15 to ~30% recovery postreperfusion. Both compounds preferentially reduced the conductance of large diameter axons. We suggest the residual ATP in our ischemic model can protect anatomic integrity and physiological functioning of spinal axons following ischemic insult. This further suggests that oxygen-glucose deprivation alone cannot be solely responsible for short-term functional and anatomic damage. The damaging effects of ischemia in vivo may be mediated by factors originating from the gray matter of the cord or other systemic factors; both were largely eliminated in our in vitro white matter preparation. axons; neurotrauma; reperfusion; mitochondria; membrane

Cell-attached recordings revealed [K.sup.+] channel activity in basolateral membranes of guinea pig distal colonic crypts. Inwardly rectified currents were apparent with a pipette solution containing 140 mM [K.sup.+]. Single-channel conductance ([Gamma]) was 9 pS at the resting membrane potential. Another inward rectifier with [gamma] of 19 pS was observed occasionally. At a holding potential of -80 mV, [Gamma] was 21 and 41 pS, respectively. Identity as [K.sup.+] channels was confirmed after patch excision by changing the bath ion composition. From reversal potentials, relative permeability of [Na.sup.+] over [K.sup.+] ([P.sub.Na]/[P.sub.K]) was 0.02 [+ or -] 0.02, with [P.sub.Rb]/[P.sub.K] = 1.1 and [P.sub.Cl]/[P.sub.K] < 0.03. Spontaneous open probability ([P.sub.o]) of the 9-pS inward rectifier ([sup.gp][K.sub.ir]) was voltage independent in cell-attached patches. Both a low ([P.sub.o] = 0.09 [+ or -] 0.01) and a moderate ([P.sub.o] = 0.41 [+ or -] 0.01) activity mode were observed. Excision moved [sup.gp][K.sub.ir] to the medium activity mode; [P.sub.o] of [sup.gp][K.sub.ir] was independent of bath [Ca.sup.2+] activity and bath acidification. Addition of [Cl.sup.-] and [K.sup.+] secretagogues altered [P.sub.o] of [sup.gp][K.sub.ir]. Forskolin or carbachol (10 [micro]M) activated the small-conductance [sup.gp][K.sub.ir] in quiescent patches and increased [P.sub.o] in low-activity patches. [K.sup.+] secretagogues, either epinephrine (5 [micro]M) or prostaglandin [E.sub.2] (100 nM), decreased [P.sub.o] of [sup.gp][K.sub.ir] in active patches. This [sup.gp][K.sub.ir] may be involved in electrogenic secretion of [Cl.sup.-] and [K.sup.+] across the colonic epithelium, which requires a large basolateral membrane [K.sup.+] conductance during maximal [Cl.sup.-] secretion and, presumably, a lower [K.sup.+] conductance during primary electrogenic [K.sup.+] secretion. chloride secretion; potassium secretion; prostaglandin [E.sub.2]; epinephrine

Legionella pneumophila is the cause of Legionnaires' disease, which is a form of potentially fatal pneumonia. To identify genes required for virulence of the bacterium, a library of 1,386 L. pneumophila signature tagged transposon mutants was studied for guinea pig virulence. The mutants were screened in pools of 96 each in a guinea pig model of L. pneumophila pneumonia. Sixteen unique mutant clones were determined to have attenuated virulence after being screened twice in the animal model. All 16 mutants failed to multiply in both lungs and spleens. Four of the sixteen had no apparent defect for intracellular multiplication in macrophages. Partial DNA sequences of the interrupted genes adjacent to the transposon insertions showed that six of them had mutations in five known L. pneumophila virulence genes: dotB, dotF/icmG, dotO/icmB, icmX, and proA. Three of the sequenced clones contained mutations in genes without known homology to other published bacterial genes, and seven clones appeared to be homologous to five different known bacterial genes but are still being characterized. With this methodology, we demonstrate the existence of L. pneumophila genes responsible for non-macrophage-related virulence. The discovery of L. pneumophila virulence genes indicates the utility of the signature tagged mutagenesis technique for pulmonary pathogens.

The perforated-patch and cell-attached mode of the patch clamp technique was used to examine the electric properties of capillary fragments isolated from guinea pig hearts. Results revealed a voltage-dependent K+ current that was activated at potentials positive to -20 mV, displayed a sigmoid rising phase and could play a role in membrane potential oscillations of the endothelium. Depolarization-activated single-channel currents that inactivated totally within 30 sec were also found in the cell-attached mode.

An experiment was performed on guinea pigs to investigate the sympathetic vasoconstriction of identified small ear arteries in response to electrical stimulation of the cervical sympathetic nerve. The roles of the cotransmitters norepinephrine (NE), ATP and neuropeptide Y (NPY) in sympathetic vasoconstriction were also examined. Results showed that NE and NPY act as cotransmitters to regulate sympathetic constriction of small ear arteries, but ATP has not role in this constriction.