Temperature-sensitive phenotype in mice lacking pituitary adenylate cyclase-activating polypeptide.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly conserved hormone. Targeted disruption of the PACAP gene has revealed a role for this peptide in lipid metabolism, carbohydrate metabolism, and the sympathetic response to insulin stress. We report here that PACAP null mice are temperature sensitive. When raised at 21 C, only 11% of the PACAP null mice survived past the first 2 wk after birth, but when raised at 24 C, most (76%) of the PACAP null mice survived. The question is the mechanism by which the absence of PACAP affects thermoregulation. Brown adipose tissue is the major site of adaptive thermogenesis in neonates and rodents. We show that PACAP null mice have brown adipocytes that differentiate normally and express two enzymes involved in thermogenesis, hormone-sensitive lipase and uncoupling protein 1. Likewise, levels of catecholamines in the adrenal medulla and plasma are normal in PACAP null mice raised at a lower temperature. In contrast, norepinephrine and its precursor dopamine extracted from brown adipose tissue are present at significantly lower levels in the PACAP null mice compared with controls. Also, PACAP null mice showed a greater loss of core body temperature compared with wild-type controls at 21 C. We conclude that under prolonged but mild cold stress, lack of PACAP results in inadequate heat production due to insufficient norepinephrine stimulation of brown adipose tissue.

5-Hydroxytryptamine is biotransformed by CYP2C9, 2C19 and 2B6 to hydroxylamine, which is converted into nitric oxide.

There is circumstantial evidence suggesting that 5-hydroxytryptamine (5-HT) could be biotransformed by enzymatic systems other than monoamino oxidase A, and that the isoforms of cytochrome P450 may be a source of nitric oxide. This study aimed to assess whether cytochrome P450 contributes to 5-HT biotransformation, and to provide evidence that 5-HT metabolism generates nitric oxide. Addition of 5-HT to cultured hepatocytes yielded 5-hydroxyindol acetic acid, a formation modulated by cytochrome P450 enzyme inducers and inhibitors. Recombinant human CYP2B6, 2C9 and 2C19 biotransformed 5-HT in 5-hydroxyindol acetic acid, but not CYP1A2, 2D6 or 3A4. Cultured hepatocytes with 5-HT generated nitric oxide, the amount of which was altered by cytochrome P450 enzyme inducers and inhibitors. In the presence of CYP2B6, 2C9 and 2C19, 5-HT relaxed precontracted isolated aortic rings, with or without endothelium, an effect prevented by the addition of methylene blue and an inhibitor of catalase, but not by myoglobin. In the absence of catalase, hydroxylamine was always assayed as a byproduct of 5-HT metabolism. In conclusion, CYP2B6, 2C9 and 2C19 biotransform 5-HT, yielding hydroxylamine, which is converted to nitric oxide in the presence of catalase. British Journal of Pharmacology (2004) 141, 407-414. doi:10.1038/sj.bjp.0705632

A new model of isolated systolic hypertension induced by chronic warfarin and vitamin K1 treatment.

BACKGROUND: Isolated systolic hypertension is the predominant form of hypertension in the elderly population. Reduction of arterial compliance appears to contribute to the elevation of pulse pressure (PP) and among potential mechanisms, gradual vascular calcification, fragmentation of elastic lamellae, and augmentation of rigid component like collagen could contribute to increase aortic stiffening. Few experimental models of the disease are currently available. METHODS: To induce large artery calcification, rats were treated with warfarin and vitamin K(1) (WK) for 4 and 8 weeks, to inhibit the maturation of matrix Gla protein. The impact of chronic PP elevation was determined on large artery and cardiac remodeling and on aortic endothelial function. RESULTS: The WK treatment led to aortic medial calcification and a proportional elevation of PP, attributable mainly to a selective elevation of systolic blood pressure. The chronic treatment also increased collagen, whereas elastin decreased in the aorta. Pulse wave velocity, an index of aortic stiffening, increased in rats treated with WK. However, indices of left ventricular and aortic hypertrophy and remodeling remained normal. In addition, the WK treatment did not modify the vasoconstriction to norepinephrine and endothelin-1, and the vasodilatory response to acetylcholine and sodium nitroprusside. CONCLUSIONS: Chronic treatment with WK represents a new model of isolated systolic hypertension with several characteristics of the human disease. The relative ease to induce calcification in this model may help to foster more fundamental research, which is lacking in this type of hypertension.

Central anti-hypertensive effect of tachykinin NK3 receptor antagonists in rat.

Tachykinins are involved in the central autonomic control of blood pressure. In the present study, we examined the i.c.v. cardiovascular effects of several tachykinin receptor antagonists in awake spontaneously hypertensive rats (SHR, 15 weeks old). Results showed that two tachykinin NK(3) receptor antagonists (R-820: 3-indolylcarbonyl-Hyp-Phg-N(Me)-Bzl and SB 222200: (S)-(-)-N-(alpha-ethylbenzyl)-3-methyl-2-phenylquinoline-4-carboxamide) caused a sustained and dose-dependent reduction of blood pressure when injected i.c.v. but not i.v. The stereoselective anti-hypertensive effect of SB 222200 peaked at 3 h and faded at 6 h post-injection (if injected at 07:00 h) or had a slower onset and peaked at 8 h post-injection (if injected at 13:00 h). The effect of R-820 was maximal at 24 h and lasted up to 48 h post-injection. Both antagonists failed to alter blood pressure in normotensive Wistar-Kyoto rats (WKY) and heart rate was not affected in both strains. The anti-hypertensive effect of SB 222200 was not associated with changes in plasma levels of catecholamines and vasopressin and it remained unchanged in SHR subjected to acute bilateral nephrectomy. In contrast, blood pressure was not affected by tachykinin NK(1) (RP 67580: (+/-) 7,7-diphenyl-2[1-imino-2(2-methoxy-phenyl)-ethyl]perhydroisoindol-4-one(3aR,7aR)) and NK(2) (SR 48968: (S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide) receptor antagonists. Data suggest that brain tachykinin NK(3) receptors are implicated in the maintenance of hypertension in SHR. Hence, these receptors may represent promising therapeutic target in the treatment of arterial hypertension.

Determination of interstitial rocuronium concentrations in the muscle tissue of anesthetized dogs by microdialysis.

INTRODUCTION: The objective was to establish and validate a microdialysis technique for the quantification of interstitial concentrations of the neuromuscular blocker, rocuronium, in the muscle tissue of dogs under steady-state conditions. METHODS: The standard and combined retrodialysis approaches were used for in vivo microdialysis probe calibration. After induction of anesthesia with pentobarbital (30 mg/kg), the left femoral vein was cannulated and blood drawn for protein binding determination. Microdialysis probes were inserted in the muscle and calibrated in vivo, using vecuronium as the calibrator. Each dog received a short 2-min infusion followed by a 120-min infusion of rocuronium via the right jugular vein and three microdialysis samples were collected at steady-state during a 2-h period. Samples were stored at -70 degrees C until HPLC analysis. RESULTS: Using combined retrodialysis, rocuronium unbound interstitial (C(ISFu)) and venous plasma (C(pssuv)) concentrations are in good agreement; with a ratio C(ISFu)/C(pssuv) of 100+/-11%. Using standard retrodialysis, this ratio was 47+/-7%. CONCLUSIONS: Combined retrodialysis is a more reliable and accurate technique for quantitative assessment of rocuronium interstitial concentrations especially for lengthy anesthetic procedures. These findings have potential implications, as drug concentrations in the site of action would be more relevant for concentration-effect relation of muscle relaxants.

Basal somatodendritic dopamine release requires snare proteins.

Dopaminergic neurons have the capacity to release dopamine not only from their axon terminals, but also from their somatodendritic compartment. The actual mechanism of somatodendritic dopamine release has remained controversial. Here we established for the first time a rat primary neuron culture model to investigate this phenomenon and use it to study the mechanism under conditions of non-stimulated spontaneous firing (1-2 Hz). We found that we can selectively measure somatodendritic dopamine release by lowering extracellular calcium to 0.5 mm, thus confirming the previously established differential calcium sensitivity of somatodendritic and terminal release. Dopamine release measured under these conditions was dependent on firing activity and independent of reverse transport through the plasma membrane. We found that treatment with botulinum neurotoxins A and B strongly reduced somatodendritic dopamine release, thus demonstrating the requirement for SNARE proteins SNAP-25 and synaptobrevin. Our work is the first to provide such direct and unambiguous evidence for the involvement of an exocytotic mechanism in basal spontaneous somatodendritic dopamine release.

Nitric oxide inhibits the bradykinin B2 receptor-mediated adrenomedullary catecholamine release but has no effect on adrenal blood flow response in vivo.

The role of nitric oxide (NO) in bradykinin (BK)-induced adrenal catecholamine secretion still remains obscure. The present study was to investigate whether an inhibition of NO synthase with N(omega)-nitro-L-arginine methyl ester (L-NAME) would modulate BK-induced adrenal catecholamine secretion (ACS) and adrenal vasodilating response (AVR) in anesthetized dogs. Plasma catecholamine concentrations were determined with an HPLC coupled with an electrochemical detector. All drugs were locally administered to the left adrenal gland via intra-arterial infusion. BK dose-dependently increased both ACS and AVR. Hoe-140, a selective B(2) antagonist, significantly blocked the BK-induced increases in both ACS and AVR. In the presence of L-NAME, the BK-induced ACS was significantly enhanced, while the simultaneous AVR remained unaffected. These results suggest that the both BK-induced ACS and AVR are primarily mediated by B(2) receptors in the canine adrenal gland. Our results also suggest that the enhanced ACS in response to BK in the presence of L-NAME may have resulted from a specific inhibition of NO formation in the adrenal gland. It is concluded that the BK-induced NO may play an inhibitory role in the B(2)-receptor-mediated mechanisms regulating ACS, while it may not be implicated in the B(2)-receptor-mediated AVR under in vivo conditions.

The cardioprotective effect of dual metallopeptidase inhibition: respective roles of endogenous kinins and natriuretic peptides.

The objective of the present study was to assess the cardioprotective effect of dual NEP-ACE inhibition in relation to endogenous cardiac bradykinin (BK), its active metabolite des-Arg9-BK, endogenous brain natriuretic peptides (BNP), and cGMP. Rats were treated with the dual metallopeptidase inhibitor, omapatrilat, or the ACE inhibitor, ramipril, for 7 d (1 mg.kg(-1).d(-1)). Hearts were then isolated and subjected to a zero-flow ischemia and reperfusion (except controls), in the absence or presence of either a B2-receptor antagonist (Hoe-140), a B1-receptor antagonist (Lys-Leu8-des-Arg9-BK), or the GC-A/GC-B-receptor antagonist (HS-142-1). Chronic omapatrilat and ramipril increased the amount of endogenous BK collected upon reperfusion, but only ramipril increased that of des-Arg9-BK. Only omapatrilat increased both peak BNP and peak cGMP upon reperfusion, those increases being blocked by Hoe-140. Chronic omapatrilat (but not ramipril) decreased the total noradrenaline and lactate dehydrogenase release during the reperfusion period. Importantly, only omapatrilat improved the functional recovery of the ischemic reperfused heart, with a reduced left ventricular end-diastolic pressure, and improved developed left ventricular pressure. All cardio protective effects of omapatrilat were blocked by Hoe-140 and by HS-142-1, but not by the B1-receptor antagonist. In conclusion, a chronic treatment with a dual metallopeptidase inhibitor demonstrated a cardioprotective action not observed with an ACE inhibitor in a context of severe ischemia in rat isolated hearts, which was mediated by both endogenous BK and BNP.

PACAP release from the canine adrenal gland in vivo: its functional role in severe hypotension.

This study was to investigate if endogenous pituitary adenylate cyclase-activating polypeptide (PACAP) can be released during direct splanchnic nerve stimulation in vivo and to determine whether PACAP in the adrenal gland can modulate the medullary response to sympathoadrenal reflex. The output of adrenal catecholamine and PACAP-38-like immunoreactivity (PACAP-38-ir) increased in a frequency-dependent manner after direct splanchnic nerve stimulation (0.2-20 Hz). Both responses were highly reproducible, and PACAP-38-ir output closely correlated with catecholamine output. Sodium nitroprusside (SNP; 0.1 mg/kg iv bolus) caused a severe hypotension resulting in marked increases in catecholamine secretion. In the presence of local PACAP-27 (125 ng), the maximum catecholamine response to SNP was significantly potentiated in a synergistic manner compared with that obtained in the group receiving SNP or PACAP-27 alone. The study indicates that endogenous PACAP-38 can be released particularly when the sympathoadrenal system is highly activated and that the local exogenous PACAP-27 enhanced the reflex-induced catecholamine release, suggesting collectively a facilitating role of PACAP as neuromodulator in the sympathoadrenal function in vivo.

Heterogeneity in the acute control of vascular protein synthesis in vivo.

In response to both hemodynamic and neurohumoral changes, the cardiovascular system remodels and this process could contribute to end organ damage. The aim of this study was to determine the early in vivo interactions between 3 systems known to contribute to vascular hypertrophic remodeling, in conduit and resistance arteries. Exogenous angiotensin II, norepinephrine and endothelin 1 administration elevated protein synthesis in the aorta and in small mesenteric arteries. In small arteries, the effect of angiotensin II was blocked by angiotensin II type 1, alpha-adrenergic and endothelin receptor antagonists, while only the alpha-adrenergic and endothelin receptor antagonists inhibited the effect of norepinephrine. Moreover, only the endothelin receptor antagonist significantly blunted the effect of exogenous endothelin on protein synthesis. In the aorta, the stimulation of angiotensin II on protein synthesis was also inhibited by the 3 antagonists. However, only the alpha-adrenoceptor antagonist blunted the response to norepinephrine, and the 3 antagonists prevented the endothelin-induced elevation of protein synthesis. The blood pressure effects of the drugs did not correlate with their capacity to stimulate or inhibit vascular protein synthesis. In conclusion, interactions in the control of protein synthesis are heterogeneous along the vascular tree. In small arteries, the interaction is linear with endothelin as the downstream effector. In the aorta, the local sympathetic nervous system appears to control protein synthesis. The heterogeneity in downstream effectors should be considered in studies investigating signaling events related to protein synthesis, which is used as an early marker of hypertrophy.

Effects of PACAP(1-27) on the canine endocrine pancreas in vivo: interaction with cholinergic mechanism.

The aim of the present study was to characterize the effects of pituitary adenylate cyclase activating polypeptide (PACAP) on the endocrine pancreas in anesthetized dogs. PACAP(1-27) and a PACAP receptor (PAC(1)) blocker, PACAP(6-27), were locally administered to the pancreas. PACAP(1-27) (0.005-5 microg) increased basal insulin and glucagon secretion in a dose-dependent manner. PACAP(6-27) (200 microg) blocked the glucagon response to PACAP(1-27) (0.5 microg) by about 80%, while the insulin response remained unchanged. With a higher dose of PACAP(6-27) (500 microg), both responses to PACAP(1-27) were inhibited by more than 80%. In the presence of atropine with an equivalent dose (128.2 microg) of PACAP(6-27) (500 microg) on a molar basis, the insulin response to PACAP(1-27) was diminished by about 20%, while the glucagon response was enhanced by about 80%. The PACAP(1-27)-induced increase in pancreatic venous blood flow was blocked by PACAP(6-27) but not by atropine. The study suggests that the endocrine secretagogue effect of PACAP(1-27) is primarily mediated by the PAC(1) receptor, and that PACAP(1-27) may interact with muscarinic receptor function in PACAP-induced insulin and glucagon secretion in the canine pancreas in vivo.