Absence Of Phentolamine Research Articles

Chronic early life stress induces alpha 1‐adrenergic receptor (α 1 ‐AR) desensitization in the renal vasculature

We have demonstrated that maternal separation (MS), a model of early life stress, induces a renal nerve-dependent reduction in glomerular filtration rate. The aim of the present study was to investigate the role of the α1-ARs in renal vasculature and cortex after MS (day 2-14 life, 3 hr/day). Tissue from adult male MS and control rats was flash frozen for α1-AR binding receptor assays and RT-PCR. Renal vasculature and cortex were homogenized for obtaining membrane preparations. Tissues were incubated with [3H]prazosin (2-12 nM) in the presence or absence of phentolamine (50 μM). Bound and free ligands were separated using a tissue harvester. Renal cortical Bmax of α1-ARs was not different between MS and C groups (40±7 vs. 47±8 fmol/mg protein; Kd 93±16 vs. 116±22 pM); however, renal vascular Bmax of α1-ARs was reduced in MS rats (4±1 vs. 39±8 mol/mg protein, p<0.05; Kd 14±6 vs. 68±12 pM). α1-ARs mRNA correlated with receptor number in renal cortex, while mRNA was increased in all 3 α1-AR subtypes in the vasculature from MS tissue compared to controls (p<0.05). Furthermore, renal nerve stimulation studies were conducted in anesthetized rats. Renal blood flow was measured while renal nerves were electrically stimulated at 1.5, 3.0, 4.0, and 6.0 Hz (1.0 ms; 1 - 8 V). The constrictor responses were attenuated in MS vs. control rats (p<.0.05). These data support the existence of a mechanism of adrenergic desensitization in response to an increased sympathetic outflow to the kidney. The extent to which α1-adrenergic peripheral vascular desensitization occurs and the mechanisms involved in intact animals is the focus of ongoing studies. Funding: R00 HL111354

Association of serum angiopoietin-like protein 2 and epinephrine levels in metabolically healthy but obese individuals: In vitro and in vivo evidence

In the present study, we explored the association of serum angiopoietin-like protein 2 (ANGPTL2) levels with insulin sensitivity and serum epinephrine levels in metabolically healthy but obese (MHO) subjects. We also investigated the effects of epinephrine on ANGPTL2 expression in adipocytes in vitro. We examined the metabolic characteristics and serum ANGPTL2 and epinephrine levels in 100 non-diabetic obese postmenopausal women. Subjects were classified as MHO (n=25) or at-risk (n=25) based on the upper and lower quartiles of insulin sensitivity, respectively. Differentiated 3T3-L1 adipocytes were treated with increasing doses of epinephrine (10, 30 and 50 nM) in the presence or absence of phentolamine (10 μM), propranolol (0.3 μM), LY294002 (50 μM) or protein kinase A inhibitor fragment 6–22 amide (PKAI, 1 mM) for 24 h. We observed that serum ANGPTL2 levels were negatively correlated with insulin sensitivity (r=−0.23, P=0.021) and serum epinephrine level (r=−0.62, P<0.001) in the study subjects, with the MHO subjects displaying significantly lower serum ANGPTL2 and higher serum epinephrine levels than the at-risk subjects. Epinephrine reduced the ANGPTL2 mRNA and protein levels in differentiated 3T3-L1 adipocytes in a dose-dependent manner. Propranolol and PKAI were able to eliminate this reduction in ANGPTL2 levels whereas phentolamine and LY294002 were not. The in vitro findings indicated that epinephrine decreased ANGPTL expression at the mRNA and protein levels via the β-adrenoceptors and the PKA signaling pathway. This study suggests that β-receptor activation helps to maintain the metabolic profile of MHO individuals and prevent type 2 diabetes mellitus (T2DM) by decreasing serum ANGPTL2 levels.

Acute Stress Reduces Wound-Induced Activation of Microbicidal Potential of Ex Vivo Isolated Human Monocyte-Derived Macrophages

BackgroundPsychological stress delays wound healing but the precise underlying mechanisms are unclear. Macrophages play an important role in wound healing, in particular by killing microbes. We hypothesized that (a) acute psychological stress reduces wound-induced activation of microbicidal potential of human monocyte-derived macrophages (HMDM), and (b) that these reductions are modulated by stress hormone release.MethodsFourty-one healthy men (mean age 35±13 years) were randomly assigned to either a stress or stress-control group. While the stress group underwent a standardized short-term psychological stress task after catheter-induced wound infliction, stress-controls did not. Catheter insertion was controlled. Assessing the microbicidal potential, we investigated PMA-activated superoxide anion production by HMDM immediately before and 1, 10 and 60 min after stress/rest. Moreover, plasma norepinephrine and epinephrine and salivary cortisol were repeatedly measured. In subsequent in vitro studies, whole blood was incubated with norepinephrine in the presence or absence of phentolamine (norepinephrine blocker) before assessing HMDM microbicidal potential.ResultsCompared with stress-controls, HMDM of the stressed subjects displayed decreased superoxide anion-responses after stress (p’s <.05). Higher plasma norepinephrine levels statistically mediated lower amounts of superoxide anion-responses (indirect effect 95% CI: 4.14–44.72). Norepinephrine-treated HMDM showed reduced superoxide anion-production (p<.001). This effect was blocked by prior incubation with phentolamine.ConclusionsOur results suggest that acute psychological stress reduces wound-induced activation of microbicidal potential of HMDM and that this reduction is mediated by norepinephrine. This might have implications for stress-induced impairment in wound healing.

In vitro determination of the mechanism of the uterine stimulatory effect of Newbouldia laevis

The uterine stimulatory effect of the ethanol leaf extract of Newbouldia laevis (Beauv.) Seemann ex Bureau (Bignoniaceae) was evaluated in the presence of some antagonists in vitro in an attempt to elucidate the mechanism of action of the extract. The extract was tested in the presence and absence of phentolamine (4.09 and 40.91 nM), diphenhydramine (4.45 and 44.47 nM), atropine (1.18 and 11.91 nM), and verapamil (2.03 and 20.35 nM). The effect of the antagonists on the extract and on oxytocin used as a reference drug in this study was evaluated. The EC50 and Emax were determined and statistically analyzed using one way ANOVA and Dunnett’s post hoc test. There was no significant difference in the EC50 and Emax of the extract and oxytocin in the presence of phentolamine. Diphenhydramine and atropine significantly inhibited (p <0.01) the extract but both drugs had no effect on oxytocin. However, significant differences (p <0.01) were observed in the EC50 and Emax of the extract and oxytocin in the presence of verapamil. These results suggest that the leaf extract of N. laevis contracts the uterus by opening voltage-operated calcium channels and/or by activation of muscarinic receptors.

Pharmacological characterization of the β-adrenoceptor that mediates the relaxant response to noradrenaline in guinea-pig tracheal smooth muscle

Pharmacological characteristics of beta-adrenoceptors (beta-ARs) mediating noradrenaline-induced relaxation were investigated in guinea-pig tracheal smooth muscle. The inhibitory effects of several types of beta-AR antagonists on noradrenaline-induced relaxation against histamine contraction were scrutinized with Schild plot analysis. The concentration-response curve for noradrenaline obtained in the absence of phentolamine and uptake inhibitors was competitively antagonized by all of the beta-AR antagonists used in this study (propranolol, bupranolol, atenolol, butoxamine and ICI-118,551). However, their pA2 values were markedly less than the expected values for beta1-AR and beta2-AR. On the other hand, pA2 values of ICI-118,551 (6.85) determined in the presence of phentolamine suggested a contribution of a beta1 -AR rather than beta2 -AR. In the presence of phentolamine and uptake inhibitors (desipramine and deoxycorticosterone), the Schild plot for atenolol was a better fit, with two distinct straight lines. The pA2 values of atenolol provided by the regression were: approximately 7.0, which corresponds to the expected beta1-AR value, and approximately 6.5, which was 3 times less than the expected value for beta1 -AR, and thus the possible presence of two classes of beta1 -AR (beta1(Low) and beta1(High)) was suggested. This view was also supported by Schild plot analysis for propranolol, which fit two straight lines each with a slope of 1.0. The present findings indicate that beta1 -ARs contributing to noradrenaline-elicited relaxation in guinea-pig tracheal smooth muscle exhibit diverse pharmacological characteristics and may be subdivided into at least two classes with distinct affinities for atenolol. Under physiological conditions, beta1(Low) rather than beta1(High) seems to play a more significant role in noradrenaline-regulated airway smooth muscle tone.

Saw palmetto is an indirectly acting sympathomimetic in the rat-isolated prostate gland

To investigate whether saw palmetto that inhibits alpha1-adrenoceptor binding in vitro affects contractility of the rat prostate gland. The effects of a commercially available saw palmetto extract were examined on the contractility of rat-isolated prostate glands. The extract was tested in the presence and absence of phentolamine, prazosin, yohimbine, propranolol, hexamethonium, cocaine, desipramine, nifedipine, guanethidine, atropine, and alpha,beta-methylene ATP to evaluate the mechanism of action. Isolated preparations of rat vas deferens and bladder were used for comparison. Unexpectedly, saw palmetto extract caused contractions of the rat prostate gland that could be attenuated by prazosin, phentolamine, nifedipine, guanethidine, cocaine, and desipramine but not by any of the other pharmacological tools. Similar contractile effects were observed in rat-isolated vas deferens preparations but not in rat-isolated bladder preparations. In the rat prostate gland, saw palmetto extract causes indirect alpha1-adrenoceptor-mediated contractions via the release of noradrenaline from sympathetic neurons.

Sympathetic control of arterial membrane potential by ATP-sensitive K(+)-channels.

Stimulation of perivascular nerve terminals leads to a release of various neurotransmitters such as norepinephrine, epinephrine, acetylcholine, nitric oxide, and calcitonin gene-related peptide (CGRP). Because some of these substances have been shown to cause smooth muscle hyperpolarization by direct or endothelium-dependent mechanisms, we hypothesized that the liberation of 1 or more of these transmitters may lead to neurogenic hyperpolarization in arterial muscle cells. The present study was designed to determine the presence or absence of neurogenic hyperpolarization and, if present, its underlying mechanisms in isolated rat mesenteric resistance arteries, through the use of conventional microelectrode techniques. The experiments were performed under the combined blockade of alpha-adrenoceptors and purinoceptors with phentolamine and suramin to eliminate depolarizing responses to nerve stimulation. Under these conditions, perivascular nerve stimulation (5 Hz, 30 seconds) evoked smooth muscle hyperpolarization (-3.3+/-0.3 mV, n=15), which was abolished by tetrodotoxin, indicating the neurogenic origin of the response. This neurogenic hyperpolarization was resistant to atropine, nitro-L-arginine, or CGRP8-37, a CGRP antagonist, but was abolished by guanethidine and beta-blocker propranolol. This hyperpolarization was also abolished by glibenclamide, an ATP-sensitive K(+) channel (K(ATP)) blocker, but was unaffected by apamin, a Ca(2+)-activated K(+) channel blocker. In separate experiments, exogenous norepinephrine caused glibenclamide-sensitive hyperpolarization in the presence of phentolamine. On the other hand, norepinephrine-induced depolarization in the absence of phentolamine was enhanced by propranolol. These findings suggest that neurally released catecholamines cause membrane hyperpolarization through the activation of K(ATP) by beta-adrenoceptors. Such hyperpolarization may play an important role in the control of arterial membrane potential by opposing alpha-adrenergic depolarization.

Effects of A1-adenosine receptor antagonists on purinergic transmission in the guinea-pig vas deferens in vitro.

1. Intracellularly recorded excitatory junction potentials (ej.ps) were used to study the effects of adenosine receptor antagonists on neurotransmitter release from postganglionic sympathetic nerve terminals in the guinea-pig vas deferens in vitro. 2. The A1 adenosine receptor antagonists, 8-phenyltheophylline (10 microM) and 8-cyclopentyl-1,3-dipropylxanthine (0.1 microM), increased the amplitude of e.j.ps evoked during trains of 20 stimuli at 1 Hz in the presence, but not in the absence, of the alpha2-adrenoceptor antagonist, yohimbine (1 microM) or the non-selective alpha-adrenoceptor antagonist, phentolamine (1 microM). 3. Adenosine (100 microM) reduced the amplitude of e.j.ps, both in the presence and in the absence of phentolamine (1 microM). This inhibitory effect of adenosine is most likely caused by a reduction in transmitter release as there was no detectable change in spontaneous ej.p. amplitudes. 4. In the presence of phentolamine, application of the adenosine uptake inhibitor, S-(p-nitrobenzyl)-6-thioinosine (0.1 microM), had no effect on ej.p. amplitudes. 5. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (100 microM), significantly increased the amplitudes of all e.j.ps evoked during trains of 20 stimuli at 1 Hz, both in the presence and in the absence of phentolamine (1 microM). 6. These results suggest that endogenous adenosine modulates neurotransmitter release by an action at prejunctional A1 adenosine receptors only when alpha2-adrenoceptors are blocked.

Effects of endothelin-1 on arterial and venous resistances in anaesthetized rats

The effects of endothelin-1 and vehicle (0.9% NaCl) on mean arterial pressure, heart rate, mean circulatory filling pressure, systemic arterial resistance, cardiac output and venous resistance were studied in four groups of pentobarbitone-anaesthetized rats, either in presence or absence of phentolamine. I.v. bolus injections of endothelin-1 at 0.5, 1 and 2 nmol/kg dose dependently increased mean arterial pressure (22, 34 and 40 mmHg), arterial resistance (33, 93 and 122% over baseline), venous resistance (40, 117 and 143% over baseline) and mean circulatory filling pressure (1.0, 1.7 and 1.8 mmHg), but decreased heart rate (−16, −21 and −17 beats/min) and cardiac output (−6, −28 and −35% below baseline). The vehicle did not significantly alter any of these variables. During the continuous infusion of phentolamine (300 μg/kg per min), endothelin-1 caused similar increases in arterial resistance, venous resistance and mean circulatory filling pressure, similar reduction in cardiac output but significantly greater pressor and bradycardic responses, suggesting that the arterial and venous constrictor effects of endothelin-1 are not due to sympathetic activation and the stimulation of α-adrenoceptors. The results show that endothelin-1 raised mean arterial pressure via the increment in systemic arterial resistance, since cardiac output was markedly reduced. This decrease in cardiac output was mediated by increases in arterial as well as venous resistances. The vasoconstrictor and venoconstrictor effects of endothelin-1 were independent of sympathetic tone.

Effects of phentolamine or yohimbine on naloxone's actions during endotoxin shock in rats.

Endogenous opioids are known to mediate some of the cardiovascular sequelae of sepsis. Inhibition of adrenergic action has been implicated as a physiological path by which endogenous opioids cause deleterious changes in cardiovascular function during endotoxin shock, but where and to what extent this accounts for changes in regional vascular resistance remains unclear. In this study, we addressed this question by examining the role of alpha-adrenergic actions in cardiovascular performance and the regional perfusion changes caused by naloxone during endotoxin shock. Rats had catheters inserted into the tail artery, left cardiac ventricle, and jugular vein. Twenty-four hours later, rats received saline or endotoxin (2 mg/kg) challenge intravenously over 30 min, followed at 40 min by intravenous naloxone (or saline) treatment (4 mg/kg + 2 mg/kg x h) in the presence or absence of phentolamine (100 micrograms/kg + 600 micrograms/kg x h) or yohimbine (40 micrograms/kg + 4 micrograms/kg x h). Radiolabeled microspheres were used to determine cardiac outputs and blood flows at 0, 30, 60, and 120 min after beginning endotoxin infusion. Naloxone attenuated the endotoxin-induced decline in mean arterial pressure (MAP) and cardiac output (CO), but had no effect on increased systemic vascular resistance (SVR). Phentolamine blocked naloxone's ability to increase MAP and CO, but permitted an increase in SVR by naloxone. In the presence of yohimbine, naloxone still increased MAP, but not CO nor SVR. Regional vascular responses varied, with naloxone demonstrating a vasoconstrictive effect despite alpha-adrenergic receptor blockade in some beds, and no effect in others. The response of individual organs in the hepatosplanchnic circulation was heterogenous as well. These data suggest that some effects of endogenous opioids during endotoxin shock are mediated via inhibition of alpha-adrenergic effects, but that some cardiovascular effects of endogenous opioids are independent of adrenergic control during endotoxin shock.

Possible participation of histamine H 3 receptors in the modulation of noradrenaline release from rat spinal cord slices

Rat spinal cord slices prelabelled with [ 3H]noradrenaline were superfused with a medium containing 1 μM desipramine plus 0.3 μM phentolamine. Histamine (0.01-10 μM) and the selective histamine H 3 receptor agonist R-(−)-α-methylhistamine (0.001-10 μM) caused a concentration-dependent decrease in the release of radioactivity evoked by electrical field stimulation (0.8 Hz, 20 mA, 2 min). The inhibitory effect of histamine was not modified by either pyrilamine (1 μM) or ranitidine (10 μM), but it was antagonized by burimamide (1 μM). The inhibitory action of histamine (1 μM) was attenuated by pertussis toxin (3 μg/ml) and was abolished by N-ethylmaleimide (30 μM). Neither forskolin (10 μM) nor rolipram (100 μM), nor the combination of both drugs, modified the inhibitory effect of histamine. Histamine (1 μM) did not modify the overflow of tritium induced by electrical stimulation in the absence of phentolamine. The present results suggest that in the rat spinal cord the release of noradrenaline elicited by electrical stimulation is negatively modulated by histamine, probably through the activation of histamine H 3 receptors. This modulatory mechanism is likely to involve the participation of regulatory G o G i proteins.

Time-course and frequency dependence of sympathetic stimulation-evoked inhibition of vagal effects at the sinus node

Vagally-induced chronotropic responses have been shown to be inhibited after the termination of sympathetic stimulation. We sought to characterize the onset and time-course of the sympathetically evoked inhibition of vagal effects by measuring vagally induced chronotropic responses during concomitant sympathetic stimulation. In anesthetized dogs we recorded lead II of the electrocardiogram, arterial pressure and cardiac cycle length. In 7 dogs, the vagi were stimulated for 15 s every minute before, during and after 10-min trains of sympathetic stimulation. The sympathetic stimulation was applied at frequencies of 0.5, 1, 2, 5 and 10 Hz. During the 1, 2 and 5-Hz trains of sympathetic stimulation, the vagally induced changes in cycle length diminished progressively and thus, were less ( P < 0.001) at 3, 5 and 10 min compared with 1 min into the sympathetic stimulation. The magnitude of the attenuation of vagal effects on cycle length depended ( P < 0.001) on the frequency of sympathetic stimulation. To determine the role of alpha- and beta-adrenergic receptors, we measured vagally-induced changes in cycle length during 5-min trains of sympathetic stimulation (1, 2, 5, 10 Hz) in the presence and absence of phentolamine and propanolol ( n = 6). Both before and after combined alpha- and beta-adrenergic receptor blockade, the vagally-induced changes in cycle length decreased ( P < 0.03) progressively during the 1, 2, 5 and 10-Hz trains of sympathetic stimulation and the magnitude of the inhibition depended ( P < 0.002) on the frequency of sympathetic stimulation. These data show that the effects of short trains of vagal stimulation on cardiac cycle length are inhibited progressively during continuous trains of sympathetic stimulation before and after combined alpha- and beta-adrenergic receptor blockade. Thus, substances other than norepinephrine may contribute to the inhibition of cardiac vagal effects that occurs during a continuous train of sympathetic stimulation.

Inhibitory Effect of Cilnidipine on Vascular Sympathetic Neurotransmission and Subsequent Vasoconstriction in Spontaneously Hypertensive Rats

We reported previously that cilnidipine inhibited increases in blood pressure and plasma norepinephrine (NE) level in response to cold stress in spontaneously hypertensive rats (SHRs). In the present study, we investigated the effect of cilnidipine on sympathetic neurotransmission and subsequent vasoconstriction in SHRs. In pithed SHRs, electrical sympathetic nerve stimulation (ESNS) elevated blood pressure, and this pressor response was abolished by guanethidine. Cilnidipine at 10 μg/kg, i.v. and phentolamine at 1 mg/kg, i.v. suppressed the pressor response to ESNS by 28±6% and 67±3%, respectively. Neither nifedipine nor nicardipine inhibited it. The pressor response to exogenous NE was not influenced by cilnidipine. α,β-Methylene ATP inhibited the pressor response to ESNS in the presence or absence of phentolamine. Cilnidipine also attenuated the phentolamine-resistant pressor response to ESNS. In SHR mesenteric vasculatures preloaded with [3H]-NE, cilnidipine (10−7 M) as well as co-conotoxin significantly inhibited the 3H overflow evoked by periarterial nerve stimulation. In radioligand binding experiments, cilnidipine inhibited [125I]-ω-conotoxin binding to rat synaptosomes, but it did not inhibit [3H]-prazosin binding to rat cortex membranes. These results suggest that cilnidipine may reduce electrically stimulated NE release from the sympathetic nerve endings of SHR vasculatures probably through its N-type Ca channel blocking action and that cilnidipine may also inhibit the vasoconstriction induced by ATP released concomitantly during nerve stimulation.

Phentolamine and hypoxia: modulation of contractility and ?1-adrenoceptors in isolated rat atria

The hypoxia-induced effects on the binding sites and affinity constant of adrenoceptors, in the presence and absence of phentolamine, were determined for atrial membranes of hearts from normal and genetically hyperlipidaemic Yoshida (YOS) rats. Atrial function was also measured during normoxia and hypoxia, in the presence and absence of phentolamine. Hypoxia increased alpha 1-adrenoceptor density in atrial membranes of normal rats (Bmax 10.6 to 26.7 fmoles/mg protein). Phentolamine prevented the increase in the Bmax of alpha 1-adrenoceptors and increased the equilibrium dissociation constant of these receptors (KD 0.17 to 0.53 nmol/l). Beta-adrenoceptors did not change during hypoxia, but the Bmax was slightly increased (26%) in the presence of phentolamine. Thus, the alpha 1/beta ratio increased from 0.40 in normoxia to 1.06 in hypoxia. In normoxic atria from YOS rats, the alpha 1/beta ratio was already elevated (0.86) in comparison to control rats (mainly due to a higher density of alpha 1-adrenoceptors in atrial membranes from YOS rats). This ratio was not modified by hypoxia (0.84), but decreased when phentolamine was present (0.30). Hypoxia reduced the force of contraction and increased diastolic tension of atria of normal rats, while the sinus rate was not significantly modified. Phentolamine abolished the increase in diastolic tension and reduced the negative effect of hypoxia on contractile force. In YOS rat atria, functional parameters were modified by hypoxia in a qualitatively similar way to that of normal rat atria.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of .ALPHA.1-Adrenoceptor Subtypes in Human Hypertrophied Prostate Using (3H)YM617, an .ALPHA.1-Selective Antagonist.

The purpose of this study was to demonstrate the distribution of α1-adrenoceptors in the human hypertrophied prostate with [3H]YM617 (a newly synthesized α1-blocker), and to determine the receptor subtype of [3H]YM617 binding sites by the use of chlorethylclonidine dihydrochloride (CEC). The slices of prostate, removed en bloc in 7 patients with benign prostatic hypertrophy and sectioned vertically to the urethra, were preincubated with or without CEC (10μM). The slices were then incubated with [3H]YM617 (1nM) in the presence or absence of phentolamine (100μM). Specific binding of [3H]YM617 was seen relatively homogeneous, and distributed in the periglandular interstitium (mainly corresponding to smooth muscles) of the prostate. The CEC-pretreated specimens showed a decrease in density of specific binding sites (30.9% mean decrease compared with the CEC non-pretreated specimens on quantitative autoradiogram) (p<0.05), but localization was almost the same as in the CEC non-pretreated specimens. Membrane assay gave similar results. These experiments indicate that ca 70% of α1-adrenoceptor binding sites in the hypertrophied prostate are insensitive to CEC, while ca 30% of that are sensitive to CEC.

Reduced Na(+)-K+ pump activity in diabetic rabbit carotid artery: reversal by aldose reductase inhibition

This study addresses the question of whether a decrease in basal Na+ pump [Na(+)-K(+)-adenosinetriphosphatase (ATPase)] activity occurs in the carotid artery of an alloxan-diabetic rabbit and, if so, whether it is associated with altered 86Rb+ uptake and contractile response to ouabain and K(+)-free solution. Ouabain-sensitive 86Rb+ uptake, an index of Na+ pump activity, was diminished approximately 50% in carotid arteries from diabetic rabbits. Concurrent with this, contractions induced by incubating the carotid arteries in a K(+)-free solution (in the absence of phentolamine) were significantly larger in the diabetic group. Readdition of K+ (1 mM) to arteries contracted with the K(+)-free solution caused relaxations that were slower to occur and of lesser magnitude in diabetic than in control rabbits. In contrast to the contractions caused by the K(+)-free medium, contractions caused by incubation with ouabain (1 mM) in the presence of phentolamine were significantly smaller in the diabetic group. Treatment of diabetic rabbits with an aldose reductase inhibitor, zopolrestat, at both high and low doses restored the alterations in vascular reactivity toward normal. The results indicate that the Na+ pump activity is diminished in the carotid artery of diabetic rabbit, and this is associated with abnormal vascular responsiveness and increased polyol pathway flux.

Na(+)-Ca2+ exchange, myoplasmic Ca2+ concentration, and contraction of arterial smooth muscle.

Na(+)-Ca2+ exchange is proposed to be an important regulator of myoplasmic intracellular Ca2+ concentration ([Ca2+]i) and contraction in vascular smooth muscle. We investigated the role of Na(+)-Ca2+ exchange in regulating [Ca2+]i in swine carotid arterial tissues that were loaded with aequorin to allow simultaneous measurement of [Ca2+]i and force. Reversal of Na(+)-Ca2+ exchange, by reduction of extracellular Na+ concentration ([Na+]o) to 1.2 mM, induced a large increase in aequorin-estimated [Ca2+]i and a low [Ca2+]i sensitivity. The contraction induced by 1.2 mM [Na+]o was partially caused by depolarization and opening of L-type Ca2+ channels because 10 microM diltiazem partially attenuated the 1.2 mM [Na+]o-induced increases in [Ca2+]i. High dose ouabain (10 microM), a putative endogenous Na+,K(+)-ATPase inhibitor, increased both [Ca2+]i and force. However, the increases in [Ca2+]i and force were mostly blocked by 10 microM phentolamine, suggesting the predominant effect of ouabain was to increase norepinephrine release from nerve terminals. In the presence of 10 microM phentolamine, 10 microM ouabain slightly accentuated 1 microM histamine-induced increases in [Ca2+]i and force. The ouabain dose necessary to induce contraction in the absence of phentolamine was significantly less than the ouabain dose necessary to accentuate histamine-induced contractions in the presence of phentolamine. These results suggest that Na(+)-Ca2+ exchange exists in swine arterial smooth muscle. These data also suggest that ouabain (which should increase [Na+]i and inhibit Na(+)-Ca2+ exchange) primarily enhances contractile function in the swine carotid artery by releasing catecholamines from nerve terminals; direct action of Na+,K(+)-ATPase inhibitors on smooth muscle appears to occur only with very high doses.

Inhibitory prejunctional muscarinic receptors at sympathetic nerves do not operate through a cyclic AMP dependent pathway

In mouse atria previously incubated with [3H]-noradrenaline, carbachol (1.0 mumo1/l) significantly inhibited the fractional stimulation-induced (S-I) outflow of radioactivity. The inhibitory effect of carbachol was greater in the presence of the alpha-adrenoceptor antagonist phentolamine (1.0 mumol/l), which by itself significantly increased the S-I outflow of radioactivity. In both cases the inhibitory effect of carbachol was blocked by atropine (0.3 mumol/l), suggesting that the effect was mediated through muscarinic receptors. 8-Bromo cyclic AMP (270 mumol/l) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 100 mumol/l), was used to maximally enhance the S-I outflow of radioactivity through the cyclic AMP mechanism. The inhibitory effect of carbachol either in the presence or in the absence of phentolamine, was not reduced in the presence of 8-bromo cyclic AMP and IBMX. Similar results with carbachol in the presence of 8-bromo cyclic AMP and IBMX were also found in rat right atrial strips which had been incubated with [3H]-noradrenaline. These results suggest that the effects through inhibitory prejunctional muscarinic receptors are not mediated by cyclic AMP. The protein kinase inhibitor, staurosporine (0.1 mumol/l), significantly blocked the enhancing effects of 8-bromo cyclic AMP (270 mumol/l) plus IBMX (100 mumol/l) on the S-I outflow of radioactivity from rat atrial strips. The inhibitory effect of carbachol (1.0 mumol/l) however, was not reduced in the presence of staurosporine, suggesting that protein kinases affected by staurosporine (protein kinase A, protein kinase C) are not involved in the post-receptor mechanism for inhibitory prejunctional muscarinic receptors. This finding further rules out the involvement of cyclic AMP in muscarinic inhibition. The inhibitory effect of carbachol either by itself or in the presence of phentolamine, was not reduced in atria from mice that had been pretreated with pertussis toxin (1.5 or 3.0 micrograms). Furthermore, in rat atrial strips, the inhibitory effect of carbachol either in the presence or in the absence of phentolamine, was also not altered by pretreating the rats with pertussis toxin (8.4 micrograms). The results suggest that in both tissues the major mechanism for inhibition of noradrenaline release through muscarinic receptors does not involve a pertussis toxin sensitive G protein.

Modulation of Adrenoceptor-Mediated Cardiovascular Effects by Short-Term In Vivo Infusion of Isoproterenol in Rats

After a 16-h in vivo infusion period of isoproterenol (400 micrograms/kg/h) from a minipump implanted subcutaneously (s.c.) in rats, we observed an increase in heart weight due to tissue edema. In isolated perfused heart preparations, the EC50s of isoproterenol to induce positive inotropy and increase in coronary flow were increased approximately 7 and 4 times, respectively. Isoproterenol dose-response curves performed in trachea preparations, were shifted to the right by about fivefold as compared with the control group. In the presence of phentolamine, the isoproterenol induced maximal relaxation in the isolated aorta from isoproterenol-pretreated animals was reduced from 46.5 to 9.2% as compared with saline-pretreated rats. In the absence of phentolamine, the epinephrine (EPI) and norepinephrine (NE) cumulative dose-response curves in this preparation were not affected by isoproterenol pretreatment with respect to EC50 and maximal effect. However, in the presence of phentolamine, the contractile response to a supramaximal dose of NE (10 microM) amounted to 30 and 70% in the saline- and isoproterenol-pretreated rats, respectively. In the presence of both phentolamine and propranolol, a similar response of 70% was observed by this supramaximal dose of agonist in the saline-pretreated rats as well. In anesthetized rats, 120 min after removal of the minipumps, sodium nitroprusside induced increase in heart rate (HR) was reduced after isoproterenol pretreatment, whereas for salbutamol the decrease in diastolic blood pressure (DBP) was also reduced. As compared with salbutamol, a marked increase was observed in the ratio of mean arterial blood pressure (MAP) to HR for SNP after isoproterenol pretreatment. The phenylephrine-induced increase in MAP was increased after isoproterenol pretreatment. We conclude that in the pathogenesis of heart failure the beta-adrenoceptor-mediated effects of catecholamines are attenuated and alpha-adrenoceptor-mediated effects become progressively important.

Phentolamine reverses NPY-induced inhibition of insulin secretion in isolated rat islets

It has been shown that the rodent pancreas is innervated by neuropeptide Y (NPY) nerves, some of which are adrenergic, and that NPY inhibits glucose-induced insulin secretion in vivo in the mouse and that from isolated rat islets in vitro. We now investigated whether the α-adrenoceptor antagonist phentolamine effects the inhibitory action of NPY on insulin secretion from isolated rat islets. It was found that NPY dose dependently inhibited insulin secretion stimulated by glucose (11.1 mM). At a concentration of 10 −7 M, NPY totally abolished the insulin secretory response to glucose. It was also found the incubation with the α-adrenoceptor antagonist phentolamine (10 −6 M) itself enhanced the insulin secretion at 3.3 mM but not at 16.7 mM glucose. Moreover, phentolamine counteracted the inhibitory action of NPY. Thus, at 10 −8 M, NPY could no longer inhibit insulin secretion when phentolamine (10 −6 M) was present, whether 3.3 mM or 11.1 mM glucose was present. In contrast, somatostatin (10 −7 M) could inhibit insulin secretion, both in the presence and absence of phentolamine (10 −6 M); this showed that phentolamine does not reverse all types of inhibition. However, when the dose of phentolamine was decreased to 10 −7 M, the inhibitory action of NPY on glucose-induced insulin secretion was retained indicating that the reversal of the NPY effect by phentolamine is a competitive effect. It is concluded (1) that NPY inhibits glucose-induced insulin secretion by a direct action on the islets, and (2) that phentolamine reverses this inhibitory action of NPY in a competitive manner.