Nitric oxide synthase (NOS) blockade increases blood pressure (BP) and modifies glomerular and tubular function. Angiotensin II (AII) blockade restores glomerular and tubular function but does not lower BP. We measured plasma renin activity (PRA), plasma (AIIp), and kidney tissue (AIIk) AII with radioimmunoassay to investigate the dissociation between renal and systemic effects of NOS blockade. Two period clearance studies followed by plasma and renal tissue harvesting were performed in seven groups of rats. Groups 1 and 1A served as controls. Groups 2 and 2A received NaCl-NaHCO3 during the first period and N(G)-monomethyl-L-arginine (L-NMMA, 0.5 mg/kg/min) during the second period. Group 3 was similar to group 2 but renal perfusion pressure (RPP) was maintained constant by using an aortic snare. Groups 4 and 4A received N(G)-nitro-L-arginine-methyl ester (L-NAME, 5 mg/100 mL of drinking water) for 2 weeks. NOS blockers decreased AIIp (group 1, 74 +/- 7 pg/mL; group 2, 22 +/- 1 pg/mL; group 3, 26 +/- 1 pg/mL; group 4, 19 +/- 3 pg/mL). The decrease in AIIp was a direct effect of L-NMMA independent of changes in perfusion pressure, as AIIp was similar in group 3 (normal RPP) and groups 2 and 4 (increased RPP). Measurements of PRA and AIIp demonstrated a similar reduction in PRA and AIIp in rats treated with NOS blocker. Although NOS blockers decreased AIIp, acute or chronic administration of NOS blockers did not modify AIIk (group 1, 1,192 +/- 51; group 2, 1,354 +/- 85; group 3, 1,348 +/- 180; group 4, 1,276 +/- 172 pg/kidney). Our findings demonstrate that NO blockers produce a dissociation between plasma and kidney AII levels. This dissociation can explain the beneficial effects of AII blockers on renal function and their lack of antihypertensive effects in anesthetized rats treated with NOS blockers.

Administration of the nitric oxide (NO) synthase blocker, NG-monomethyl L-arginine prevents the increase in glomerular filtration rate (GFR) normally observed with glycine, an effect that is restored by angiotensin II (All) blockers. These findings suggest that changes in NO and All dictate the presence or absence of renal vasodilation during amino acid (AA) infusion. We examined the effect of branched-chain (BCAA) and non-branched-chain (NBCAA) AA on GFR, NO, and All to determine if abnormal NO or All responses could explain the absence of vasodilation with BCAA. Our findings demonstrated that NBCAA increased GFR and NO and did not modify All, either plasma (Allp) or kidney (Allk) All. The response with BCAA was strikingly different. L-Valine increased GFR without modifying NO or All. L-Leucine increased Allk and NO but did not increase GFR. Administration of All blockers (captopril or losartan) was associated with an increase in GFR during infusion of leucine. Single nephron studies demonstrated that increased Allk with L-leucine was associated with decreased absolute proximal reabsorption and probably activation of the tubuloglomerular feedback. An AA-specific increase in Allk is critical to inhibition of the normal renal response to AA infusion. NO generation is an important mediator but not the sole mechanism that determines the increase in GFR during amino acid infusion.

Intracerebroventricular (ICV) injection of carbachol elicits hormonal and metabolic responses similar to moderate stress. In normal dogs, ICV carbachol stimulated marked counterregulatory hormone release, but altered plasma glucose only marginally because the marked increment in glucose production (Ra) was almost matched by the increment of utilization (Rd), even though plasma insulin was unchanged. In alloxan-diabetic dogs, Rd did not match Ra and plasma glucose increased substantially. Since somatostatin octapeptide (ODT8-SS) inhibits some sympathetic mechanisms of the stress response, we explored the extent to which ODT8-SS can alleviate the counterregulatory responses to stress induced by carbachol, and particularly whether it can restore glycemic control in diabetes. ODT8-SS (20 nmol) was ICV-injected (1) in normal dogs (n = 5), and (2) prior to ICV carbachol before (n = 7) and after (n = 6) the induction of alloxan-diabetes. ODT8-SS did not affect basal values, but when administered before ICV carbachol there were no significant increments in plasma epinephrine, cortisol, arginine vasopressin (AVP), insulin, glucose, or lactate. There were significant increases in norepinephrine, glucagon, Ra, Rd, and the glucose metabolic clearance rate (MCR), although they were much smaller than seen previously with ICV carbachol alone. After induction of alloxan-diabetes, Rd and MCR did not change with ICV ODT8-SS and carbachol as in normal dogs, but norepinephrine, epinephrine, glucagon, lactate, plasma glucose, and Ra increased, although with the exception of glucagon these increases were much smaller than seen previously with ICV carbachol alone. ODT8-SS administered before ICV carbachol in normal or diabetic animals resulted in increased free fatty acid (FFA) levels. The increases in glycerol were less than and those in FFA greater than seen previously with ICV carbachol alone. Since ODT8-SS does not alter basal counterregulatory hormone release but suppresses the release during stress, this is a useful probe to analyze some of the metabolic responses to stress. When the response to carbachol from our previous report is compared with the responses to carbachol + ODT8-SS, it is indicated that the stress-related increase in Ra was consistent with stimulation of the sympathetic nervous system, whereas increased Rd is related to an unknown stress-related neuroendocrine mechanism that requires a permissive effect of insulin, since it was not seen in the frankly diabetic animals. We hypothesize that the stress-induced increase in Rd occurs not only in muscle but also in adipocytes, and that the somatostatin-induced attenuation of Rd decreased FFA re-esterification and consequently markedly increased stress-induced FFA release. Although ODT8-SS substantially decreased the Ra response in diabetic dogs, the hyperglycemic effect of carbachol is only partially attenuated. Thus, in diabetes moderate stress can disturb glucose homeostasis substantially even when the sympathetic nervous system has been markedly suppressed.

To further elucidate the minimum bioactive conformation of neuropeptide Y (NPY), a series of truncated and conformationally constrained analogues has been prepared. The synthesis and purification of these peptides was achieved using routine laboratory strategies and techniques. Parent molecules consisted of the native NPY N-terminal 1-4 and C-terminal 25-36 segments, having the residue 5-24 core replaced by either a single flexible omega-aminoalkanoic acid, or a more rigid Pro-Gly or Pro-DAla sequence which was expected to constrain a putative turn, and allow the N- and C-termini to align. Cross-linking between residues 2 and 27 through lactamization using side-chain length and chirality suggested by computer simulations, resulted in cyclo-(2/27)-des-AA7-24[Glu2,Gly6,DDpr27]NPY that exhibited very high affinity (Ki = 0.3 versus 0.3 nM for NPY) for the Y2 receptor using SK-N-BE2 human neuroblastoma cells, yet very low affinity for the Y1 receptor using SK-N-MC human neuroblastoma cells (Ki = 130 versus 2.0 nM for NPY). The added constraint resulting from bridging in this analogue as well as in others suggested that the combination of the deletion of residues 5-24 and the introduction of an internal ring produced exclusive selectivity for the Y2 receptor with little or no loss of affinity. The tolerance of structural recognition was further demonstrated as a second ring was introduced which was expected to constrain the amphiphilic alpha-helix, resulting in the full Y2 agonist dicyclo (2/27,28/32)-des-AA7-24 [Glu2,32,DAla6,DDpr27,Lys28]NPY. Improvement of Y1 binding activity was achieved only by including more residues (des-AA10-17) in the central PP-fold region, while allowing limited flexibility of the termini. Although the length of the bridge seemed to have little effect on binding potency, changes in the location of and chirality at the bridgehead resulted in analogues with different binding affinities. Combination of optimum structural modifications resulted in cyclo-(7/21)-des-AA10-18[Cys7,21]NPY, an analogue shortened by 25% but retaining comparable binding properties to that of native NPY at Y1 and Y2 receptor types (Ki = 5.1 and 1.3 nM, respectively).

Although studies indicate that converting enzyme inhibitors such as captopril influence beta-adrenergic physiology, the data on alpha-adrenergic physiology is inconsistent. This study therefore examined the effects of captopril (50 mg/day for 5 days) during sodium restriction on the pressor response and on angiotensin II and neuropeptide Y levels to infused norepinephrine (0.01 to 0.1 mug/kg/min) in 17 hypertensive and 27 normotensive subjects. Angiotensin II increased significantly in response to infused norepinephrine during placebo administration (p < 0.001) but not during captopril administration (p = 0.15). Neuropeptide Y levels decreased in response to captopril (p = 0.02). Despite these changes the pressor response to infused norepinephrine was unchanged with captopril. These data support the conclusion that the antihypertensive action of captopril is unrelated to alterations in norepinephrine-mediated alpha-adrenergic pressor regulation. The finding of a decrease in neuropeptide Y levels may have relevance to the therapeutic effects of captopril.

Corticotropin-releasing hormone (CRH) acts at the pituitary level to increase ACTH secretion and, within the central nervous system, to stimulate the sympathoadrenomedullary axis and behavioral activity. In addition, the central administration of CRH has been reported to reduce cellular immunity in the periphery. This study investigated the temporal relationship between CRH receptor regulation and the changes in splenic natural killer (NK) cell and pituitary-adrenocortical hormone responses to a single intracisternal (IC) CRH challenge (acute CRH) 24 h after chronic CRH pretreatment (5 nmol/day IC CRH for 4 days). Chronic CRH significantly decreased by 44.2 +/- 7.8% (P < 0.01) the CRH receptor concentration (beta max) in the amygdala. In contrast, the CRH receptor concentration of the anterior pituitary in the chronic CRH group was similar to the pituitary CRH receptor concentration in chronic saline controls. The immunoreactive-CRH concentration of the amygdala measured 24 h after the last IC CRH injection was not different from brain CRH levels in controls receiving chronic saline pretreatment. Consequently, the downregulation of amygdala CRH receptors occurred after transient increases in intracerebral CRH levels and did not result from ex vivo receptor occupancy by residual exogenous CRH sequestrated in brain tissue at the time of the CRH binding assay. Pretreatment with chronic CRH completely abolished the ability of a central CRH injection to suppress splenic NK activity; whereas, pituitary-adrenal responses to a superimposed acute CRH challenge were not significantly altered by chronic CRH pretreatment. These results suggest that the desensitization of the brain processes mediating CRH-induced suppression of splenic NK cytotoxicity is temporally correlated with CRH receptor downregulation in the amygdala but independent of pituitary-adrenal activation. These findings represent the first in vivo demonstration of homologous downregulation of extrahypothalamic CRH receptors and provide further evidence for the role of central CRH in the modulation of immune function.

OBJECTIVES:The purpose of this study was to examine the effects of short-term captopril therapy during sodium restriction on several markers of the sympathetic nervous system, including plasma norepinephrine, neuropeptide Y, beta-adrenergic receptors and cortisol.BACKGROUND:Recent studies suggest that the therapeutic effects of converting enzyme inhibitors involve not only the renin-angiotensin and prostaglandin systems but also the sympathetic system.METHODS:Twelve hypertensive and 20 normotensive men were studied after 2 5-day hospital stays during which they consumed a 10-mEq sodium diet and received captopril (25 mg twice daily) or placebo in a double-blind crossover study.RESULTS:Captopril decreased neuropeptide Y (p < 0.05) and angiotensin II (p < 0.01) and increased isoproterenol-stimulated cyclic adenosine monophosphate (AMP) in lymphocytes (p < 0.03), plasma norepinephrine (p < 0.02), cortisol (p < 0.05) and renin (p < 0.001) in both hypertensive and normotensive subjects. Hypertensive subjects had an increased beta-adrenergic receptor density (p < 0.02) and a greater decrease in diastolic blood pressure compared with normotensive subjects (p < 0.02).CONCLUSIONS:The results of this study suggest that the short-term therapeutic effects of captopril may involve concerted changes in key components of the sympathetic nervous system. These findings, such as decreased neuropeptide Y combined with increased norepinephrine and beta-adrenergic receptors, are compatible with the observation of increased cardiac output and decreased peripheral resistance after short-term angiotensin-converting enzyme inhibition.

Glycine (G) infusion causes renal vasodilation mediated by nitric oxide (NO). Cyclosporine A (CsA) nephrotoxicity is characterized by preglomerular vasoconstriction and decreased efferent arteriolar tone probably related to reduced NO and angiotensin II, respectively. L-Arginine (ARG) is a precursor to NO. To test the hypothesis that chronic CsA decreases renal NO activity, we compared the glomerular hemodynamic response to glycine infusion in rats after 8 d of CsA (30 mg/kg per d s.c.), CsA and ARG (1.6 g/kg per d p.o.) (A/CsA), and in two groups of pair-fed controls (CON, A/CON). Single nephron GFR (SNGFR), single nephron plasma flow (SNPF), glomerular capillary hydrostatic pressure gradient (delta P), proximal tubular reabsorption (APR), and kidney tissue angiotensin II (AIIk) were measured before and during G. CsA was associated with baseline decrements in SNGFR, SNPF, delta P, and AIIk, and with a blunted hemodynamic response to G. In CON, ARG did not affect baseline hemodynamics or modify the response to G. In CsA, ARG decreased baseline preglomerular resistance and restored the glomerular hemodynamic response to G. G was associated with a significant increase in AIIk in both CON and CsA. These findings suggest that (a) CsA is associated with decreased AIIk, and (b) CsA may diminish NO activity within the kidney, and that this capacity may be partially restored by arginine feeding.

It has been shown that NPY and select C-terminal fragments of NPY that evoke a hypotensive response upon intraarterial administration in the rat also cause mast cell degranulation and histamine release in vitro. Additionally, elevation of plasma histamine levels has been observed concomitant with the hypotensive effect of NPY and various C-terminal fragments. In order to investigate whether the hypotensive response to NPY18-36 is correlated to this observed elevation of histamine in vivo, we sought to characterize the structural requirements for each activity. We conducted a systematic replacement of each amino acid in NPY18-36 by its D-isomer. Additionally, various modifications were made to the N- or C-terminii of NPY18-36. The following rank order of potency was obtained for the hypotensive action of these analogues of NPY18-36 relative to NPY18-36. Only one analogue ([D-Tyr21]NPY18-36) exhibited significantly enhanced potency. Eleven analogues of NPY18-36, ([D-Thr32]-, [D-Arg35]-, [D-Ile31]-, [D-Leu30]-, [D-Tyr27]-, [D-Ser22]-, [D-Tyr36]-, [D-Gln34]-, [D-Asn29]-, [D-Ala23]-, and [D-Arg33]NPY18-36) were equipotent with NPY18-36. Four analogues ([D-His26]-, [D-Ile28]-, and [D-Ala18]NPY18-36 and -NPY18-27) had reduced potency (10-80%) while eight analogues ([D-Arg19]-, [D-Tyr20], [D-Leu24]-, [D-Arg25]-, [Ac-Ala18]-, [Me-Ala18]-, [desamino-Ala18]NPY18-36 and NPY18-36 free acid) failed to produce a significant hypotensive response (< 10%) at the doses tested. The sensitivity of NPY18-36 to chiral inversion of single residues or other modifications at the N-terminus suggested the presence of a conformationally well defined N-terminal pharmacophore. Additionally, five NPY18-36 analogues were tested for elevation of plasma histamine levels. The rank order of potency ([D-Thr32]NPY18-36 = [D-Tyr21]NPY18-36 >> NPY18-36 > [D-Ala18]NPY18-36 > [Ac-Ala18]NPY18-36) was correlated with each analogue's potency at evoking a hypotensive response. In contrast, NPY1-36 failed to evoke an elevation in plasma histamine levels despite its hypotensive effects. Hence, we conclude that the magnitude of the hypotensive response evoked by an NPY18-36 analogue is primarily a function of its ability to elevate plasma histamine levels. However, the mechanism underlying NPY1-36-evoked hypotension appears to be different.

The neuroendocrine response to electroconvulsive therapy (ECT) was assessed in four patients after pretreatment with esmolol (1.0 mg/kg), fentanyl (1.5 mu;g/kg), labetalol (0.3 mg/kg), and saline solution (control). Each patient received each drug pretreatment using a double-blind, randomized study block-design. During each of the five studies, blood samples were obtained from each patient before anesthetic induction, before ECT shock, and at 1, 5, 10, and 30 min after seizure. Samples were subsequently analyzed for epinephrine, norepinephrine, adrenocorticotrophic hormone (ACTH), arginine vasopressin (AVP), and cortisol. Electroconvulsive therapy after saline pretreatment resulted in a 3-fold and 15-fold increase in norepinephrine and epinephrine levels, respectively (P < 0.05). The ACTH and cortisol levels gradually increased over 30 min, peaking at values that were two to three times the control values (P < 0.05). The AVP levels increased significantly after induction of ECT (P < 0.005) and remained higher than control levels at 5, 10, and 30 min. The effect of pretreatments varied. Pretreatment with esmolol and fentanyl resulted in significant attenuation of the norepinephrine peak after seizure (P < 0.05). Only esmolol significantly attenuated ECT-induced epinephrine secretion, whereas fentanyl pretreatment significantly reduced release of ACTH after ECT. No pretreatment significantly affected the elevated AVP or cortisol levels seen on emergence or up to 30 min after treatment. The ability of esmolol pretreatment to attenuate serum catecholamine release after ECT is consistent with its ability to block the cardiovascular responses to ECT.

SUMMARY1. A sensitive and specific neuropeptide Y (NPY) radio‐immunoassay has been developed. This radio‐immunoassay does not detect the NPY‐related peptides pancreatic polypeptide or peptide YY. NPY extracted from rat plasma using sequential C18 sorbent and affinity chromatography co‐eluted with synthetic rat NPY when applied to high pressure liquid chromatography.2. The procedure of stabilization of platelets followed by high speed centrifugation reduced basal values of NPY by 60%, and this may be consistent with removal of platelets that release NPY. Administration of the cholinesterase inhibitor physostigmine (0.3 mg/kg), intravenously, produced a small but significant increase (39%) from basal concentrations of NPY.3. NPY concentrations in young (2‐3‐month‐old) Sprague‐Dawley and Fisher 944 rats were similar; however, NPY concentrations were significantly increased (55%) in 2‐year‐old Fisher 944 rats. Similar to plasma concentrations of noradrenaline, NPY levels increase with age.

Bombesin-like immunoreactivity has been localized within neuronal cell bodies of the hypothalamus and nerve terminals within the dorsal vagal complex. The possibility that the hypothalamus is a source for bombesin-like immunoreactive terminals within the dorsal vagal complex was examined using the combined retrograde tracing and immunohistochemical technique. After injections of retrograde tracer were made into the dorsal vagal complex, cells in the hypothalamus labeled with both retrograde tracer and bombesin immunoreactivity were localized in the parvocellular part of the paraventricular nucleus. In the paraventricular nucleus most of the vagal projecting bombesin immunoreactive neurons were located within the medial parvocellular subdivision. Approximately 30% of the bombesin immunoreactive neurons in this subnucleus projected to the dorsal vagal complex. The results suggest that the paraventricular hypothalamic nucleus is a major source of bombesin terminals within the dorsal vagal complex. This pathway may mediate some of the autonomic nervous system changes that are observed when bombesin is injected within the central nervous system. Additionally, this data adds to a growing amount of evidence supporting the role of bombesin as a peptide neurotransmitter.

Studies were performed in conscious unrestrained rats to compare the ability of the CRF receptor antagonist, alpha-helical CRF9-41, to inhibit the actions of CRF in three in vivo bioassay systems. When both peptides were administered intracerebroventricularly, an antagonist:agonist ratio between 6:1-12:1 was required to abolish CRF-induced elevations of plasma catecholamine levels. When both peptides were administered iv, CRF-induced hypotension and tachycardia were completely prevented by an antagonist:agonist ratio of 6:1, whereas total blockade of CRF-induced elevations of plasma ACTH and beta-endorphin levels required an antagonist:agonist ratio of 3000:1. These results demonstrate marked differences in the ability of alpha-helical CRF9-41 to antagonize various biological actions of CRF and support the existence of multiple CRF receptor subtypes.

To identify the central nervous system site of action of bombesin to elevate plasma concentrations of catecholamines, this peptide has been injected into numerous brain ventricular and parenchymal sites. Low doses of bombesin (1–10 ng) injected into the region of the rostral nucleus tractus solitarius (NTS) elicited an elevation of plasma catecholamines greater than those observed following an injection of bombesin into other brain regions. Bombesin-induced (10 ng) elevation of plasma epinephrine but not norepinephrine was prevented by co-administration of somatostatin-28 (100 ng). Mean arterial pressure (MAP) and heart rate (HR) were measured following injection of bombesin into the NTS. Bombesin injected into the NTS resulted in prolonged decreases in HR without significantly altering MAP. These studies demonstrate that bombesin injected into the dorsal medulla resulted in significant changes of plasma catecholamine levels and HR. Based on these actions of bombesin and the neuroanatomic distribution of bombesin-like peptide, it is suggested that this peptide may play an important role in regulation of sympatho-adrenal and cardiac functions.

The neuroendocrine response to electroconvulsive therapy (ECT) was assessed in four patients after pretreatment with esmolol (1.0 mg/kg), fentanyl (1.5 mu;g/kg), labetalol (0.3 mg/kg), and saline solution (control). Each patient received each drug pretreatment using a double-blind, randomized study block-design. During each of the five studies, blood samples were obtained from each patient before anesthetic induction, before ECT shock, and at 1, 5, 10, and 30 min after seizure. Samples were subsequently analyzed for epinephrine, norepinephrine, adrenocorticotrophic hormone (ACTH), arginine vasopressin (AVP), and cortisol. Electroconvulsive therapy after saline pretreatment resulted in a 3-fold and 15-fold increase in norepinephrine and epinephrine levels, respectively (P < 0.05). The ACTH and cortisol levels gradually increased over 30 min, peaking at values that were two to three times the control values (P < 0.05). The AVP levels increased significantly after induction of ECT (P < 0.005) and remained higher than control levels at 5, 10, and 30 min. The effect of pretreatments varied. Pretreatment with esmolol and fentanyl resulted in significant attenuation of the norepinephrine peak after seizure (P < 0.05). Only esmolol significantly attenuated ECT-induced epinephrine secretion, whereas fentanyl pretreatment significantly reduced release of ACTH after ECT. No pretreatment significantly affected the elevated AVP or cortisol levels seen on emergence or up to 30 min after treatment. The ability of esmolol pretreatment to attenuate serum catecholamine release after ECT is consistent with its ability to block the cardiovascular responses to ECT.

Neuropeptide Y (NPY) is a 36-amino-acid, C-terminal amidated peptide that is found in bulbospinal pathways and can inhibit the release of the primary afferent C-fiber neurotransmitter, substance P. Based on these observations, the present studies examined the possible antinociceptive effects of this peptide and several NPY fragments after intrathecal administration in rats prepared with chronic intrathecal catheters. In the 52 degrees C hot plate test, NPY produced a dose-dependent elevation in the nociceptive threshold with a median effective dose of 1.1 nmol. The ordering of fragments' activity was: NPY greater than NPY16-36 greater than or equal to NPY19-36 greater than or equal to NPY14-36 greater than or equal to NPY18-36 much greater than NPY1-36-OH = NPY18-36-OH = 0. In the paw pressure test, NPY was not active, even at the highest doses examined (median effective dose greater than 20 nmol), whereas the C-terminal fragments retained their potency and produced significant increases in the pressure required to evoke escape (NPY18-36: median effective dose = 18.7 nmol). The rank ordering of activity in the paw pressure test was: NPY19-36 greater than or equal to NPY14-36 greater than or equal to NPY18-36 greater than or equal to NPY16-36 much greater than NPY = NPY18-36-OH = 0. Peptide YY, human pancreatic polypeptide and avian pancreatic polypeptide behave similarly to NPY.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies were performed in conscious unre- strained rats to compare the ability of the CRF receptor antag- onist, a-helical CRF9_4i, to inhibit the actions of CRF in three in vivo bioassay systems. When both peptides were administered intracerebroventricularly, an antagonist:agonist ratio between 6:1-12:1 was required to abolish CRF-induced elevations of plasma catecholamine levels. When both peptides were admin- istered iv, CRF-induced hypotension and tachycardia were com- pletely prevented by an antagonist:agonist ratio of 6:1, whereas total blockade of CRF-induced elevations of plasma ACTH and /3-endorphin levels required an antagonist:agonist ratio of 3000:1. These results demonstrate marked differences in the ability of a-helical CRF9-41 to antagonize various biological actions of CRF and support the existence of multiple CRF receptor subtypes. {Endocrinology 129: 1312-1316,1991)