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Hydroxyproline (Hyp) metabolism is a key source of glyoxylate production in the body and may be a major contributor to excessive oxalate production in the primary hyperoxalurias where glyoxylate metabolism is impaired. Important gaps in our knowledge include identification of the tissues with the capacity to degrade Hyp and the development of model systems to study this metabolism and how to suppress it. The expression of mRNA for enzymes in the pathway was examined in 15 different human tissues. Expression of the complete pathway was identified in liver, kidney, pancreas, and small intestine. HepG2 cells also expressed these mRNAs and enzymes and were shown to metabolize Hyp in the culture medium to glycolate, glycine, and oxalate. [(18)O]- and [(13)C(5)]Hyp were synthesized and evaluated for their use with in vitro and in vivo models. [(18)O]Hyp was not suitable because of an apparent tautomerism of [(18)O]glyoxylate between enol and hydrated forms, which resulted in a loss of isotope. [(13)C(5)]Hyp, however, was metabolized to [(13)C(2)]glycolate, [(13)C(2)]glycine, and [(13)C(2)]oxalate in vitro in HepG2 cells and in vivo in mice infused with [(13)C(5)]Hyp. These model systems should be valuable tools for exploring various aspects of Hyp metabolism and will be useful in determining whether blocking Hyp catabolism is an effective therapy in the treatment of primary hyperoxaluria.

The aim of this study was to compare the systemic hemodynamic effects of four commonly used anesthetic regimens in mice that were chronically instrumented for direct and continuous measurements of cardiac output (CO). Mice (CD-1, Swiss, and C57BL6 strains) were instrumented with a transit-time flow probe placed around the ascending aorta for CO measurement. An arterial catheter was inserted into the aorta 4 or 5 days later for blood pressure measurements. After full recovery, hemodynamic parameters including stroke volume, heart rate, CO, mean arterial pressure (MAP), and total peripheral resistance were measured with animals in the conscious state. General anesthesia was then induced in these mice using isoflurane (Iso), urethane, pentobarbital sodium, or ketamine-xylazine (K-X). The doses and routes of administration of these agents were given as required for general surgical procedures in these animals. Compared with the values obtained for animals in the conscious resting state, MAP and CO decreased during all anesthetic interventions, and hemodynamic effects were smallest for Iso (MAP, -24 +/- 3%; CO, -5 +/- 7%; n = 15 mice) and greatest for K-X (MAP, -51 +/- 6%; CO, -37 +/- 9%; n = 8 mice), respectively. The hemodynamic effects of K-X were fully antagonized by administration of the alpha(2)-receptor antagonist atipamezole (n = 8 mice). These results indicate that the anesthetic Iso has fewer systemic hemodynamic effects in mice than the nonvolatile anesthetics.

Glycyl-glutamine (Gly-Gln; beta-endorphin(30-31)) is an endogenous dipeptide that is synthesized through the posttranslational processing of beta-endorphin in brain stem regions that control respiration and autonomic function. This study tested the hypothesis that Gly-Gln administration to conscious rats will prevent the respiratory depression caused by morphine without affecting morphine antinociception. Rats were administered Gly-Gln (1-100 nmol) or saline (10 microl) intracerebroventricularly followed, 5 min later, by morphine (40 nmol icv). Arterial blood gases and pH were measured immediately before Gly-Gln and 30 min after morphine injection. Gly-Gln pretreatment inhibited morphine-induced hypercapnia, hypoxia, and acidosis significantly. The response was dose dependent and significant at Gly-Gln doses as low as 1 nmol. In contrast, Gly-Gln (1-300 nmol) had no effect on morphine-evoked antinociception in the paw withdrawal test. When given alone to otherwise untreated animals, Gly-Gln did not affect nociceptive latencies or blood gas values. These data indicate that Gly-Gln inhibits morphine-induced respiratory depression without compromising morphine antinociception.

Mice lacking AT(1A) receptors for ANG II have a defect in urinary concentration manifested by an inability to increase urinary osmolality to levels seen in controls after thirsting. This defect results in extreme serum hypertonicity during water deprivation. In the basal state, plasma vasopressin levels are similar in wild-type controls and Agtr1a -/- mice. Plasma vasopressin levels increase normally in the AT(1A) receptor-deficient mice after 24 h of water deprivation, suggesting that the defect in urine concentration is intrinsic to the kidney. Using magnetic resonance microscopy, we find that the absence of AT(1A) receptors is associated with a modest reduction in the distance from the kidney surface to the tip of the papilla. However, this structural abnormality seems to play little role in the urinary concentrating defect in Agtr1a -/- mice since the impairment is largely reproduced in wild-type mice by treatment with an AT(1)-receptor antagonist. These studies demonstrate a critical role for the AT(1A) receptor in maintaining inner medullary structures in the kidney and in regulating renal water excretion.

We previously demonstrated that mRen-2 transgenic [Tg(+)] rats are sensitive to chronic high NaCl intake, showing increased arterial pressure and vasopressin (VP) secretion. In this study, we determined the effect of a chronic osmotic challenge, 4 days of drinking 2% NaCl, on direct arterial blood pressure, heart rate, fluid-electrolyte balance, circadian rhythm of mean arterial pressure (MAP), and changes in plasma VP and catecholamines. Under baseline conditions, male Tg(+) rats showed a significant shift in the peak in circadian MAP into the light portion of the day-night cycle. Substitution of 2% NaCl for drinking water caused a rapid increase in MAP, 20 +/- 5 mmHg in Tg(+) rats within 6 h. Whereas the amplitude of circadian MAP fluctuations increased in salt-loaded Tg(+) rats, there was no significant change in the circadian timing of peak MAP with salt loading. Tg(+) rats showed exaggerated osmotic-induced increases in plasma VP, norepinephrine (NE), and epinephrine (Epi) compared with Tg(-) rats. Plasma NE and Epi were increased two- and fourfold, respectively, in the hypertensive rats with no significant change in the Tg(-) rats. Intravenous administration of a VP antagonist did not alter arterial pressure in either Tg(+) or Tg(-) rats. Tg(+) and Tg(-) rats showed a positive sodium balance with no significant difference observed between the groups. Tg(+) rats showed a significant increase in salt consumption, plasma sodium, osmolality, and hematocrit, accompanied by a negative water balance. We conclude that Tg(+) rats are sensitive to acute and chronic osmotic stimuli in terms of blood pressure, fluid-electrolyte balance, and plasma VP and catecholamines. Whereas elevated plasma VP does not contribute to the hypertensive response, increased sympathetic drive may mediate the salt-induced blood pressure changes in this model.

We have previously demonstrated that mRen-2 transgenic [Tg(+)] rats show a salt-induced exacerbation of hypertension (Callaha, M., P. Li, C. M. Ferrario, D. Ganten, and M. Morris. Hypertension Dallas 27: 573-577, 1996). In this study, we examined the role of paraventricular (PVN) angiotensin type-1 (AT1) receptors in the salt sensitivity of this model. Male Tg(+) and Tg(-) rats were instrumented with PVN cannulas for intracerebral drug administration and carotid catheters for chronic cardiovascular monitoring. Substitution of 2% NaCl for drinking water for 4 days caused a significant elevation (23 mmHg) of mean arterial pressure (MAP) in Tg(+) rats but not in Tg(-) rats. PVN injection of AT1 receptor antisense oligodeoxynucleotides (ASODN), but not scrambled oligodeoxynucleotides (SCODN), produced a rapid decrease in MAP of 24 +/_ 8 mmHg in salt-treated Tg(+) rats. There was no effect of either AT1 ASODN or SCODN on MAP in salt-loaded Tg(-) rats or in Tg(+) rats consuming tap water. Salt loading significantly increased subfornical organ AT1 receptors in Tg(+) rats with no changes produced by ASODN or SCODN. In contrast, there was a 40% decrease in PVN AT1 receptors 20 h after direct PVN injection of AT1 ASODN injection, compared with SCODN in Tg(+) rats. We conclude that PVN AT1 receptors are critical in the expression of salt sensitivity in mRen-2 transgenic rats.

The effects of lesioning of the anteroventral third ventricle (AV3V) region on vasopressin (VP) release into the supraoptic nucleus (SON) and blood in response to central and systemic osmotic stimulation were determined. Microdialysis probes were implanted bilaterally within the SON of male urethan-anesthetized rats with sham or AV3V lesions. Osmotic stimuli were administered intraperitoneally (3.5 M NaCl, 600 microliters/100 g body wt) and then via the microdialysis probes (1 M NaCl-artificial cerebrospinal fluid). AV3V lesions attenuated the response to systemic osmotic stimulation. The lesioned rats showed no increase in intranuclear VP release and reduced plasma VP (increase of 42.6 +/- 8.4 vs. 78.0 +/- 16.4 pg/ml) and blood pressure responses (7.1 +/- 2.3 vs. 19.6 +/- 3.2 mmHg) to intraperitoneal NaCl. In contrast, the endocrine and cardiovascular responses to direct osmotic stimulation of the nucleus were as seen in previous studies and seemed to be unaffected by the lesion. These results show that lesion of the AV3V region interrupts neuronal inputs which trigger VP secretion from the posterior pituitary as well as release into the extracellular compartment of the SON.

The effect of hypertonic NaCl consumption on vasopressin (VP) and oxytocin (OT) mRNA levels and plasma and pituitary peptides was evaluated in rats with sham or anterior ventral third ventricular (AV3V) lesions. Rats were given tap water or 2% NaCl for 4 days. Because the rats with lesions drank significantly less salt solution than the controls (78.8 +/- 17.4 vs. 205.5 +/- 37.8 ml/4 days), a second control group was included in which saline intake was matched to the lesioned group. AV3V rats showed a deficit in the peptide response to the osmotic stimulus. There was no increase in plasma VP or OT or decrease in posterior pituitary peptide content in the face of an extreme hypernatremia: plasma sodium of 180.1 +/- 4.2 meq/l. Evaluation of mRNA changes by means of in situ hybridization showed that animals with lesions responded to the salt challenge with increases in hypothalamic VP and OT mRNA levels. There were significant increases in paraventricular and supraoptic OT mRNA and paraventricular VP mRNA in the lesioned group. The salt-matched control group showed no changes in peptide mRNA levels. These results demonstrate that AV3V lesions produce an impairment of the salt-neuroendocrine reflex but a persistence of the peptide mRNA response. Differences in control mechanisms must account for this dissociation between peptide mRNA expression and peptide secretion.

Experiments were performed to determine the effect of direct osmotic stimulation of the supraoptic nucleus (SON) on central and peripheral vasopressin (AVP) release and arterial pressure. A microdialysis method was used to deliver hyperosmotic NaCl, mannitol or urea bilaterally into the SON and to sample SON extracellular fluid and blood. Simultaneous brain and blood microdialysis showed that hyperosmotic NaCl increased central and peripheral AVP release and increased mean arterial pressure (MAP). The pressor response was not blocked by intravenous injection of a V1-receptor antagonist, D(CH2)5Tyr(Me)AVP, suggesting that circulating AVP was not involved in that response. Hyperosmotic mannitol or urea caused an increase in central peptide release, but failed to affect MAP or peripheral AVP release. The results suggest that central AVP release within the SON may be due to osmoreceptor stimulation while the peripheral effects on AVP release and MAP are specific for sodium. The results also demonstrate the utility of brain and blood microdialysis for the delivery of stimuli into specific brain regions with simultaneous monitoring of central and peripheral peptide release.

The anterior ventral region of the third ventricle (AV3V) is a major central site in the regulation of cardiovascular and renal function. To examine the role of the central nervous system in the regulation of atrial natriuretic peptide (ANP) secretion, the effect of lesions in this region on basal, volume-stimulated, and osmotic-stimulated plasma ANP was determined. Basal levels of plasma ANP were not different in sham- and AV3V-lesioned rats. Volume expansion with a continuous infusion of saline or with a bolus administration of saline increased plasma ANP in sham-lesioned rats. In AV3V-lesioned rats, continuous infusion of saline had no effect on plasma ANP, and a bolus administration of saline decreased plasma ANP. Osmotic stimulation with hypertonic saline increased plasma ANP in sham-lesioned rats but had no effect on plasma ANP in AV3V-lesioned rats. These results suggest that the central nervous system is involved in the regulation of ANP secretion and that altered ANP regulation may contribute to the cardiovascular and renal deficiencies in AV3V-lesioned rats.

Examination of the cardiovascular effects produced by peripheral administration of peptide sequences derived from adrenocorticotropic hormone (ACTH) led to the discovery of the pressor, cardioaccelerator, and natriuretic actions of intravenous (iv) ACTH-(4-10). Based on pharmacological studies in rats with alpha- and beta-adrenergic receptor antagonists, the cardiovascular effects of this peptide appeared to be mediated by the release of catecholamines. A peptide sequence analogous to ACTH-(4-10), gamma-melanocyte-stimulating hormone (gamma-MSH), possesses greater than 100-fold more cardiovascular activity and 1,000-fold more natriuretic activity than ACTH-(4-10). The pressor effect of iv gamma-MSH peptides appears to be dependent on the maintenance of preganglionic sympathetic drive, with no significant contribution of circulating vasopressin or angiotensin II. However, the presence of central vasopressinergic, and perhaps angiotensinergic, pathways appears to be crucial for expression of the full pressor effect of circulating gamma-MSH. Further evidence for the potential importance of the central nervous system (CNS) in these cardiovascular effects was obtained from central lesion experiments and a comparison of intracarotid vs. intrajugular infusions. Structure-activity studies suggested that the cardiovascular effects of ACTH-(4-10) or gamma-MSH are dependent on an Arg-hydrophobic amino acid sequence, located at or near their COOH-terminal. A similar requirement for biological activity is found in molluscan cardioexcitatory peptides, and the molluscan peptides have cardiovascular effects in rats, which resemble ACTH-(4-10) or gamma-MSH. This suggests that peptides of the gamma-MSH family are the pharmacological analogues, and perhaps the physiological homologues, of a cardioexcitatory family of peptides found in molluscs and birds. Elevated circulating levels of peptides derived from the NH2-terminal of pro-opiomelanocortin (POMC) have been found in psychological stress, cardiovascular distress, and hemorrhage. Increases in central sympathetic drive are common to all of these states. gamma-MSH peptides have been localized to POMC neurons in the arcuate nucleus and nucleus commissuralis of the rat. Projections from the latter nucleus innervate hindbrain vasomotor centers. Intraventricular administration of gamma-MSH produces prolonged elevation of mean arterial pressure. gamma-MSH peptides may provide a link between humoral and neurogenic mechanisms in cardiovascular regulation and could potentially be important neurotransmitters for central control of the cardiovascular system.

Evidence from numerous laboratories has shown that administration of adrenocorticotropic hormone (ACTH) to rats produces hypertension within 5 days. However, the analysis of blood pressure in these studies was by the tail-cuff technique, an acute and indirect approach. We have now administered ACTH, via a subcutaneous depot injection (5 or 10 U/day for 9 days), to chronically instrumented rats maintained in metabolic cages. Although tail-cuff measurements of arterial pressure indicated that the ACTH treatment produced hypertension, this was not confirmed by direct 24-h measurements of mean arterial pressure. There was no effect of ACTH on 24-h heart rate throughout the treatment period compared with saline-injected controls. We also examined coefficient of variation of all our measurements. None of the factors was altered by ACTH administration. However, ACTH treatment did produce a diuretic effect, further confirming previous work and providing renal, in addition to cardiovascular, evidence for the bioavailability of the ACTH depot. These results demonstrate that chronic ACTH treatment does not produce a true hypertensive state in rats but rather may enhance the cardiovascular response to the stress of the indirect arterial pressure measurement technique.

The role of central nervous system arginine vasopressin (AVP) and oxytocin (OXY) in the cardiovascular response to acute stress was examined using three experimental models: pharmacological antagonism of central AVP-OXY receptors; lesions of the paraventricular nucleus (PVN); and rats genetically lacking in AVP synthesis, i.e., the Brattleboro strain. Central administration of an AVP-OXY antagonist abolished the increase in heart rate (HR) seen following acute footshock stress. The group receiving centrally administered antagonist increased HR 15 +/- 17 (SE) beats/min, whereas, in contrast, the group receiving intravenous administration of the antagonist showed a 66 +/- 17 beats/min increase, and the group receiving intraventricular antagonist vehicle showed a 101 +/- 14 beats/min increase in response to stress. In a second study, electrolytic lesions of the PVN also blocked the increase in HR seen following stress, 20 +/- 12 beats/min for PVN-lesioned rats, 74 +/- 25 beats/min for sham lesion rats, and 93 +/- 7 beats/min for rats with a lesion not destroying the PVN. In the final study, the responses of Brattleboro rats, i.e., rats genetically deficient in vasopressin synthesis, were equivalent to their Long-Evans controls (131 +/- 13 and 147 +/- 12 beats/min, respectively). In each of these studies, the blood pressure responses to the stressor were equivalent for control and experimental groups. The results of these studies suggest that a neuropeptide system originating in or passing through the PVN may play an important role in the cardiovascular responses to stress and further suggest that the central OXY system may be one pathway mediating this response.