Modification of cardiac sodium channels by carboxyl reagents. Trimethyloxonium and water-soluble carbodiimide

In TTX-sensitive nerve and skeletal muscle Na+ channels, selective modification of external carboxyl groups with trimethyloxonium (TMO) or water-soluble carbodiimide (WSC) prevents voltage-dependent Ca2+ block, reduces unitary conductance, and decreases guanidinium toxin affinity. In the case of TMO, it has been suggested that all three effects result from modification of a single carboxyl group, which causes a positive shift in the channel's surface potential. We studied the effect of these reagents on Ca2+ block of adult rabbit ventricular Na+ channels in cell-attached patches. In unmodified channels, unitary conductance (gamma Na) was 18.6 +/- 0.9 pS with 280 mM Na+ and 2 mM Ca2+ in the pipette and was reduced to 5.2 +/- 0.8 pS by 10 mM Ca2+. In contrast to TTX-sensitive Na+ channels, Ca2+ block of cardiac Na+ channels was not prevented by TMO; after TMO pretreatment, gamma Na was 6.1 +/- 1.0 pS in 10 mM Ca2+. Nevertheless, TMO altered cardiac Na+ channel properties. In 2 mM Ca2+, TMO-treated patches exhibited up to three discrete gamma Na levels: 15.3 +/- 1.7, 11.3 +/- 1.5, and 9.8 +/- 1.8 pS. Patch-to-patch variation in which levels were present and the absence of transitions between levels suggests that at least two sites were modified by TMO. An abbreviation of mean open time (MOT) accompanied each decrease in gamma Na. The effects on channel gating of elevating external Ca2+ differed from those of TMO pretreatment. Increasing pipette Ca2+ from 2 to 10 mM prolonged the MOT at potentials positive to approximately -35 mV by decreasing the open to inactivated (O-->I) transition rate constant. On the other hand, even in 10 mM Ca2+ TMO accelerated the O-->I transition rate constant without a change in its voltage dependence. Ensemble averages after TMO showed a shortening of the time to peak current and an acceleration of the rate of current decay. Channel modification with WSC resulted in analogous effects to those of TMO in failing to show relief from block by 10 mM Ca2+. Further, WSC caused a decrease in gamma Na and an abbreviation of MOT at all potentials tested. We conclude that a change in surface potential caused by a single carboxyl modification is inadequate to explain the effects of TMO and WSC in heart. Failure of TMO and WSC to prevent Ca2+ block of the cardiac Na+ channel is a new distinction among isoforms in the Na+ channel multigene family.     

Activation of 5-HT1A receptors in the medullary raphe reduces cardiovascular changes elicited by acute psychological and inflammatory stresses in rabbits

The present strategy for the prevention of excessive sympathetic neural traffic to the heart relies on the use of beta-blockers, drugs that act at the heart end of the brain-heart axis. In the present study, we attempted to suppress cardiac sympathetic nerve activity by affecting the relevant cardiomotoneurons in the brain using the selective serotonin-1A (5-HT(1A)) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). In conscious, unrestrained rabbits, instrumented for recordings of heart rate, arterial pressure, or cardiac output, we provoked increases in cardiac sympathetic activity by psychological (loud sound, pinprick, and air jet) or inflammatory (0.5 microg/kg iv lipopolysaccharide) stresses. Pinprick and air-jet stresses elicited transient increases in heart rate (+50 +/- 7 and +38 +/- 4 beats/min, respectively) and in mean arterial pressure (+16 +/- 2 and +15 +/- 3 mmHg, respectively). Lipopolysaccharide injection caused sustained increases in heart rate (from 210 +/- 3 to 268 +/- 10 beats/min) and in arterial pressure (from 74 +/- 3 to 92 +/- 4 mmHg). Systemically administered 8-OH-DPAT (0.004-0.1 mg/kg) substantially attenuated these responses in a dose-dependent manner. Drug effects were prevented by a selective 5-HT(1A) receptor antagonist, WAY-100635 (0.1 mg/kg iv). Similarly to systemic administration, microinjection of 8-OH-DPAT (500 nl of 10 mM solution) into the medullary raphe-parapyramidal region caused antitachycardic effects during stressful stimulation and during lipopolysaccharide-elicited tachycardia. This is the first demonstration that activation of 5-HT(1A) receptors in the medullary raphe-parapyramidal area causes suppression of neurally mediated cardiovascular changes during acute psychological and immune stresses.     

Reflex cardiovascular and ventilatory responses to increasing H+ activity in cat hindlimb muscle

Static exercise increases arterial pressure, heart rate, and ventilation, effects which are believed in part to arise reflexly from a metabolic stimulus in the working muscle. In anesthetized cats, we tested the hypothesis that intra-arterial injections of lactic and hydrochloric acid, which created levels of these substances in muscle similar to those seen during contraction, reflexly increased cardiovascular and ventilatory function. Hydrochloric acid (32 and 57 mM; 1 ml) injected into the arterial supply of the triceps surae decreased intramuscular pH from 7.26 +/- 0.05 to 7.17 +/- 0.05 (P less than 0.01) and reflexly increased arterial pressure (23 +/- 7 mmHg; P less than 0.01), heart rate (11 +/- 2 beats/min; P less than 0.001), and ventilation (187 +/- 72 ml/min; P less than 0.05). Static contraction of the triceps surae decreased intramuscular pH from 7.28 +/- 0.06 to 7.13 +/- 0.06 (P less than 0.01). Lactic acid was more potent in causing reflexes than was equimolar HCl. For example, lactic acid containing 4 mM lactate and 0.87 mM H+ reflexly increased arterial pressure, heart rate, and ventilation, whereas 0.87 mM HCl did not. Intra-arterial sodium lactate (13 and 33 mM) at a neutral pH had no effect on these variables. We conclude that contraction-induced accumulation of H+, especially that arising from lactic acid, might provide a metabolic stimulus to evoke reflex autonomic effects.     

Influence of low extracellular Ca2+ ions on the cardiac function changes induced by verapamil in the perfused rabbit heart

We tested the hypothesis that the myocardial effects of verapamil (VER) could be enhanced by decreasing the extracellular Ca2+ concentration ([Ca2+]o) in the isolated rabbit heart at 37 degrees C. After perfusion with standard Krebs - bicarbonate solution containing 1.27 mM Ca2+, for a 30-min period of stabilization and 15 min of control, groups of hearts were perfused for an additional 60 min with solutions containing one of the following: 1.27 mM Ca2+ (control group), 0.23 mM Ca2+ (low [Ca2+]o group), 1.27 mM Ca2+ plus 10(-7) M VER (VER group), or 0.23 mM Ca2+ plus 10(-7) M VER (combination, CBN group). These concentrations of [Ca2+]o and VER produce submaximal responses in our preparation. We found that the heart rate - LV pressure product (RPP) in the CBN group fell rapidly to 0 in the first 2-3 min of perfusion, this response being significantly lower than in the other two groups for the first 15 min. Electromechanical dissociation (EMD) appeared in one of six hearts at 60 min and in four of six hearts at 30 min in the low [Ca2+]o and VER groups, respectively, whereas it occurred in the CBN group in all hearts at 3 min. Depolarization rate (DR) fell by 10% in the low [Ca2+]o and VER groups versus a reduction of 45% in the CBN group (P less than 0.05) during the last 45 min of perfusion. The PR interval increased by 300% in the CBN group, a much greater and significant change (P less than 0.05) than in the hearts exposed to VER or low [Ca2+]o.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hemodynamic effects of calcitonin gene-related peptide in conscious rats

The cardiovascular effects of calcitonin gene-related peptide (CGRP) were examined in conscious, unrestrained rats. Changes in mean arterial pressure, heart rate and cardiac output were continuously monitored before and after i.v. bolus injection of CGRP (0.1-5 micrograms/kg). Injection of the peptide caused dose-dependent reductions in mean arterial pressure (-24 +/- 4 mmHg), which were accompanied by marked tachycardia. Cardiac output was significantly increased after CGRP but little change was observed in stroke volume. CGRP also reduced total peripheral resistance (-46 +/- 6%). These data indicate that the hypotensive actions of CGRP are mediated through peripheral vasodilation rather than through reductions in cardiac output. Pretreatment with propranolol significantly reduced the tachycardia responses to CGRP from 81 +/- 11 beats/min to 36 +/- 4 beats/min, but did not abolish the increase in heart rate. These data suggest that CGRP produces a tachycardia through reflex increases in cardiac sympathetic tone and through possible direct positive chronotropic effects on the heart.     

Abolition of clonidine's effects on ventricular refractoriness by naloxone in the conscious dog

The interaction between opiate and adrenergic receptors on cardiac electrophysiologic function in the conscious dog was addressed in our study. We examined the effects of opiate receptor blockade with naloxone on clonidine-induced changes in refractoriness of the cardiac ventricle. Nine dogs were chronically instrumented for recording mean arterial blood pressure, administration of drugs and for measurement of effective refractory period of the ventricle. Clonidine (10 micrograms/kg, i.v.) significantly (p less than 0.05) decreased heart rate to 72 +/- 5 beats/minute from 108 +/- 8 beats/minute; mean arterial pressure decreased significantly (p less than 0.05) to 83 +/- 3 mmHg from 91 +/- 4 mmHg. Ventricular refractoriness was increased significantly (p less than 0.05) at current levels of 7 and 10 mA and pacing rates 180 and 200 beats/minute. Naloxone (3-10 mg/kg, i.v.) abolished clonidine's effects on heart rate, mean arterial pressure and ventricular refractoriness. We conclude that ventricular refractoriness may be regulated in part by interactions between central adrenergic and opioidergic systems.     

Cardiovascular effects of urotensin II in different brain areas

It has been shown that intracerebroventricular injection of urotensin II (UII)-induced hypotensive and bradycardiac responses. Here, we tested the cardiovascular roles of UII in different brain areas by microinjection of UII into the A1 and A2 areas (noradrenergic cells found in the lower part of the medulla that have been designated either A1 or A2 areas), the paraventricular and the arcuate nucleus. In urethane-anaesthetized rats, we observed that: (1) microinjection of UII into the A1 area induced dose-related depressor and bradycardiac responses; (2) mean arterial blood pressure (mABP) and heart rate (HR) did not change significantly after microinjection of UII into the A2 area; and (3) significant increases in mABP and HR were induced after microinjection of 10 pmol UII into either the paraventricular or arcuate nucleus. The above results suggest that UII, in different brain areas, plays different roles in cardiovascular regulation and the A1 area is a very important action site for UII in cardiovascular regulation.     

Triiodothyronine concomitantly inhibits calcium overload and postischemic myocardial stunning in diabetic rats.

Acute effects of triiodothyronine (T3) on postischemic myocardial stunning and intracellular Ca2+ contents were studied in the isolated working hearts of streptozotocin-induced diabetic rats and age-matched controls. After two weeks of diabetes, serum T3 and T4 levels were decreased to 62.5% and 33.9% of control values. Basal preischemic cardiac performance did not differ between diabetic and control rats. In contrast, during reperfusion after 20-min ischemia, diabetic rats exhibited an impaired recovery of heart rate (at 30-min reperfusion 57.5% of baseline vs. control 88.5%), left ventricular (LV) systolic pressure (44.1% vs. 89.5%), and cardiac work (23.1% vs. 66.0%). When 1 and 100 nM T3 was added before ischemia, heart rate was recovered to 77.2% and 81.8% of baseline, LV systolic pressure to 68.3% and 81.9%, and cardiac work to 50.8% and 59.0%, respectively. Diabetic rat hearts showed a higher Ca2+ content in the basal state and a further increase after reperfusion (4.96+/-1.17 vs. control 3.78+/-0.48 micromol/g, p<0.01). In diabetic hearts, H+ release was decreased after reperfusion (5.24+/-2.21 vs. 8.70+/-1.41 mmol/min/g, p<0.05). T3 administration caused a decrease in the postischemic Ca2+ accumulation (lnM T3 4.66+/-0.41 and 100 nM T3 3.58+/-0.36) and recovered the H+ release (lnM T3 16.2+/-3.9 and 100 nM T3 11.6+/-0.9). T3 did not alter myocardial O2 consumption. Results suggest that diabetic rat hearts are vulnerable to postischemic stunning, and T3 protects the myocardial stunning possibly via inhibiting Ca2+ overload.     

Attenuation of corticotropin releasing factor-induced hypotension in anesthetized rats with the CRF antagonist, alpha-helical CRF9-41; comparison with effect on ACTH release.

The effect of pretreatment with the corticotropin releasing factor (CRF-41) antagonist, alpha-helical CRF(9-41), on the hypotensive response obtained on peripheral administration of CRF-41 has been assessed in anesthetized Wistar rats. A single IV bolus dose of rat CRF-41 (2 nmol, at 0 min) produced a hypotensive effect which was rapid in onset (-52 mmHg at +1 min) and sustained throughout the 60-min study period (-42, -40, -26 and -16 mmHg at +3, +10, +30 and +60 min, respectively). The antagonist [alpha CRF(9-41)] was administered in consecutive bolus doses of 12.5, 25 and 50 nmol at -15, -10 and -5 min, respectively. This had no effect on mean arterial blood pressure (MABP) or heart rate, nor did it change significantly the magnitude of the initial rapid fall in MABP when CRF-41 was administered (-45 mmHg at +1 min). However, following pretreatment with alpha CRF(9-41), MABP returned to control values within 3 min and the sustained period of hypotension was completely blocked. Administration of CRF-41 resulted in 44% and 142% increases in norepinephrine and epinephrine measured at +60 min. Pretreatment with the antagonist attenuated the rise in circulating catecholamine levels observed after CRF-41 administration. In comparison, pretreatment with the antagonist did not alter the ACTH response to CRF-41 at +1 and +3 min and only reduced ACTH levels by 28% (p less than 0.05), 43% (p less than 0.001) and 41% (p less than 0.01) at 10, 30 and 60 min, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiovascular responses to melanocortin 4-receptor stimulation in conscious unrestrained normotensive rats

In the present studies, we used a non-selective melanocortin MC3/4 receptor agonist (HP228) and a novel selective melanocortin MC4 receptor (MC4-R) agonist (MK-cpd1) to study the cardiovascular, temperature, locomotor and feeding responses to melanocortin receptor stimulation in comparison to sibutramine in rats instrumented with a telemetry transmitter. Moreover, norepinephrine turnover rates in heart and brown adipose tissue were determined. HP228 (1, 3 and 10mg/kg, i.p.) reduced 24h food intake dose-dependently and increased heart rate and mean arterial pressure (maximal differences: +60+/-8beats/min and +8+/-1mmHg, means+/-S.E.M., p<0.001 and p<0.01, respectively). After 10mg/kg HP228 showed a three-fold increase in norepinephrine turnover in the heart. The selective MC4-R agonist MK-cpd1 tended to decrease 24h food intake only at the highest dose tested (10mg/kg, i.p., p=0.06) and increased both heart rate (+17+/-4 and +22+/-5beats/min at 3 and 10mg/kg, p<0.01) and mean arterial pressure (+4+/-1mmHg at 10mg/kg, p<0.05). Sibutramine reduced food intake at all doses tested (1, 3 and 10mg/kg, i.p.). It did not change mean arterial pressure significantly, and increased heart rate only at the highest dose tested (+36+/-6beats/min, p<0.05). If also observed in humans, the pharmacological profile of MC4-R agonists would not offer a significant therapeutic advantage over currently used appetite suppressants such as sibutramine.     

Pulmonary vascular tone is increased by a voltage-dependent calcium channel potentiator

The mechanism of hypoxia-induced pulmonary vasoconstriction remains unknown. To explore the possible dependence of the hypoxic response on voltage-activated calcium (Ca2+) channels, the effects of BAY K 8644 (BAY), a voltage-dependent Ca2+ channel potentiator, were observed on the pulmonary vascular response to hypoxia of both the intact anesthetized dog and the perfused isolated rat lung. In six rat lungs given BAY (1 X 10(-6)M), hypoxia increased mean pulmonary arterial pressure (Ppa) to 30.5 +/- 1.7 (SEM) Torr compared with 14.8 +/- 1.2 Torr for six untreated rat lungs (P less than 0.01). After nifedipine, the maximum Ppa during hypoxia fell 14.1 +/- 2.4 Torr from the previous hypoxic challenge in the BAY-stimulated rats (P less than 0.01). BAY (1.2 X 10(-7) mol/kg) given during normoxia in seven dogs increased pulmonary vascular resistance 2.5 +/- 0.3 to 5.0 +/- 1.2 Torr X 1(-1) X min (P less than 0.05), and systemic vascular resistance 55 +/- 4.9 to 126 +/- 20.7 Torr X 1(-1) X min (P less than 0.05). Systemic mean arterial pressure rose 68 Torr, whereas Ppa remained unchanged. Administration of BAY during hypoxia produced an increase in Ppa: 28 +/- 1.5 to 33 +/- 1.9 Torr (P less than 0.05). Thus BAY, a Ca2+ channel potentiator, enhances the hypoxic pulmonary response in vitro and in vivo. This, together with the effect of nifedipine on BAY potentiation, suggests that increased Ca2+ channel activity may be important in the mechanism of hypoxic pulmonary vasoconstriction.     

Frequency-dependent blockade of T-type Ca2+ current by efonidipine in cardiomyocytes

Efonidipine is a dihydropyridine Ca2+ antagonist with inhibitory effects on both L-type and T-type Ca2+ channels and potent bradycardiac activity especially in patients with high heart rate. In the present study, we examined the frequency dependence of efonidipine action on the T-type Ca2+ channel in isolated guinea-pig ventricular myocytes. The potency of efonidipine to inhibit the T-type Ca2+ current was higher under higher stimulation frequencies. The IC50 values were 1.3 x 10(-8), 2.0 x 10(-6) and 6.3 x 10(-6) M under stimulation frequencies of 1, 0.2 and 0.05 Hz, respectively. The reduction of T-type Ca2+ current amplitude was not accompanied by change in the time course of current decay. Efonidipine (10 microM) inhibited T-type Ca2+ current elicited by depolarization from holding potentials ranging from -90 to -30 mV by about 30%; the voltage-dependence of steady-state inactivation was not changed by the drug. Efonidipine slowed the recovery from inactivation following an inactivating prepulse. In conclusion, efonidipine was shown to have frequency-dependent inhibitory effects on the T-type Ca2+ channel, which could be explained by slow dissociation of the drug from the inactivated state of the channel.     

Calcium shifts accompanying rest-dependent phenomena in atrial muscle of guinea-pig heart

Calcium shifts accompanying rest and post-rest phenomena in isolated left atrial appendages of guinea-pig heart were investigated by means of isotope 45Ca2+. Experiments were performed under conditions of full equilibration of preparations with isotope-containing solution (at least 45 min) in order to investigate the changes in content of exchangeable Ca, or at short exposure to isotope (2-5 min) in order to measure the excitation-dependent Ca2+ influx. Atria stimulated at the rate of 60/min for 55 min in radioactive solution contained 3.79 +/- 0.21 mM of 45Ca2+/kg of wet weight (w.w.). The rested preparations contained 2.61 +/- 0.23 mM/kg w.w. When the previously stimulated for 45 min atria were allowed to rest for 10 min, their content of 45Ca2+ dropped to 3.01 +/- 0.17 mM/kg w.w. despite continued exposure to this isotope. The first post-rest contraction was by 50% +/- 25% stronger than the control steady-state beats. Contractile force (CF) decreased during the two following beats to 25% of control. Calcium-45 content dropped during these beats to 2.50 +/- 0.14 mM/kg w.w. whereas influx of 45Ca2+ amounted to 0.24 mM/kg w.w. CF recovered to control values during 10 min of the following stimulation. Calcium-45 content reached at this time 4.09 +/- 0.12 mM/kg w.w. It is proposed that there are two intracellular store compartments in guinea-pig atrial muscle. The capacity of one of them is rate-dependent and its Ca2+ is lost at rest. This Ca2+ is partially trapped by the other compartment (presumably sarcoplasmic reticulum) and is released to activate the strong post-rest contraction.     

Comparative effects of levosimendan, OR-1896, OR-1855, dobutamine, and milrinone on vascular resistance, indexes of cardiac function, and O2 consumption in dogs

Levosimendan enhances cardiac contractility via Ca(2+) sensitization and induces vasodilation through the activation of ATP-dependent K(+) and large-conductance Ca(2+)-dependent K(+) channels. However, the hemodynamic effects of levosimendan, as well as its metabolites, OR-1896 and OR-1855, relative to plasma concentrations achieved, are not well defined. Thus levosimendan, OR-1896, OR-1855, or vehicle was infused at 0.01, 0.03, 0.1, and 0.3 mumol.kg(-1).30 min(-1), targeting therapeutic to supratherapeutic concentrations of total levosimendan (62.6 ng/ml). Results were compared with those of the beta(1)-agonist dobutamine and the phosphodiesterase 3 inhibitor milrinone. Peak concentrations of levosimendan, OR-1896, and OR-1855 were 455 +/- 21, 126 +/- 6, and 136 +/- 6 ng/ml, respectively. Levosimendan and OR-1896 produced dose-dependent reductions in mean arterial pressure (-31 +/- 2 and -42 +/- 3 mmHg, respectively) and systemic resistance without affecting pulse pressure, effects paralleled by increases in heart rate; OR-1855 produced no effect at any dose tested. Dobutamine, but not milrinone, increased mean arterial pressure and pulse pressure (17 +/- 2 and 23 +/- 2 mmHg, respectively). Regarding potency to elicit reductions in time to peak pressure and time to systolic pressure recovery: OR-1896 > levosimendan > milrinone > dobutamine. Levosimendan and OR-1896 elicited dose-dependent increases in change in pressure over time (118 +/- 10 and 133 +/- 13%, respectively), concomitant with reductions in left ventricular end-diastolic pressure and ejection time. However, neither levosimendan nor OR-1896 produced increases in myocardial oxygen consumption at inotropic and vasodilatory concentrations, whereas dobutamine increased myocardial oxygen consumption (79% above baseline). Effects of the levosimendan and OR-1896 were limited to the systemic circulation; neither compound produced changes in pulmonary pressure, whereas dobutamine produced profound increases (74 +/- 13%). Thus levosimendan and OR-1896 are hemodynamically active in the anesthetized dog at concentrations observed clinically and elicit cardiovascular effects consistent with activation of both K(+) channels and Ca(2+) sensitization, whereas OR-1855 is inactive on endpoints measured in this study.     

Comparative studies on the hypotensive effect of LHRH antagonists in anesthetized rats

Certain neuropeptides are known to cause a hypotensive response, thought to be due to mast cell degranulation. The effects of five antagonists of luteinizing hormone-releasing hormone on blood pressure and heart rate were compared in the anesthetized rat. When given intravenously, all five compounds induced hypotensive and bradycardiac effects. The order of potency for these effects was Nal-Arg Antagonist approximately detirelix [( N-Ac-D-Nal(2)1, D-pCl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10]LHRH) greater than [N-Ac-D-Nal(2)1, D-pCl-Phe2,D-Pal(3)3,D-hArg(Et2)6,L-hArg (Et2)8,D-Ala10]LHRH (RS-26306) approximately antide greater than [N-Ac-D-Nal(2)1, D-pCl-Phe2,D-Pal(3)3,6, L-hArg(Et2)8,D-Ala10]LHRH (RS-15378) and did not parallel the order of antiovulatory potencies of these compounds. The hypotensive activity of LHRH antagonists, therefore, appeared dissociable from their antiovulatory activity. RS-26306 and RS-15378 appeared to have the greatest therapeutic ratios.     

Effect of anoxia on intracellular ATP, Na+i, Ca2+i, Mg2+i, and cytotoxicity in rat hepatocytes.

The effects of anoxia were studied in freshly isolated rat hepatocytes maintained in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB). Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular sodium (Na+i) with SBFI, intracellular pH (pHi) with BCECF, lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate, ATP by 31P NMR spectroscopy in real time, and intracellular free Mg2+ (Mg2+i) from the chemical shift of beta-ATP relative to alpha-ATP in the NMR spectra. Anoxia was induced by perfusing the cells with KHB saturated with 95% N2, 5% CO2. After 1 h of anoxia, beta-ATP fell 66%, and 85% after 2 h, while the Pi/ATP ratio increased 10-fold from 2.75 to 28.3. Under control conditions, the resting cytosolic free calcium was 127 +/- 6 nM. Anoxia increased Ca2+i in two distinct phases: a first rise occurred within 15 min and reached a mean value of 389 +/- 35 nM (p less than 0.001). A second peak reached a maximum value of 1.45 +/- 0.12 microM (p less than 0.001) after 1 h. During the first hour of anoxia, Na+i increased from 15.9 +/- 2.4 mM to 32.2 +/- 1.2 mM (p less than 0.001), Mg2+i doubled from 0.51 +/- 0.05 to 1.12 +/- 0.01 mM (p less than 0.001), and pHi decreased from 7.41 +/- 0.03 to 7.06 +/- 0.1 (p less than 0.001). LDH release doubled during the first hour and increased 6-fold during the second hour of anoxia. Upon reoxygenation, ATP, Ca2+i, Mg2+i, Na+i, and LDH returned near the control levels within 45 min. To determine whether the increased LDH release was related to the rise in Ca2+i, and whether the increased Ca2+i was caused by Ca2+ influx, the cells were perfused with Ca(2+)-free KHB (+ 0.1 mM EGTA) during the anoxic period. After 2 h of anoxia in Ca(2+)-free medium, beta-ATP again fell 90%, but Ca2+i, after the first initial peak, fell below control levels, and LDH release increased only 2.7-fold. During reoxygenation, Ca2+i, ATP, Na+i, and LDH returned near the control levels within 45 min. These results suggest that the rise in Ca2+i induced by anoxia is caused by an influx of Ca2+ from the extracellular fluid, and that LDH release and cell injury may be related to the resulting rise in Ca2+i.     

Diastolic pressure determines autonomic responses to pressure perturbation in humans

Arterial baroreceptors reflexly regulate sympathetic and heart rate responses to alteration of blood pressure. The primary mechanical determinant of arterial baroreceptor activity in humans remains unclear. We examined the influence of systolic, diastolic, pulse, and mean arterial pressures on efferent muscle sympathetic nerve activity (MSNA, microneurography) and heart rate responses during perturbation of arterial pressure in 10 normal human subjects [age 25 +/- 2 (SE) yr]. We directly measured arterial pressure, heart rate, and MSNA during intravenous vasodilator infusion (nitroprusside, 6 +/- 1 micrograms.kg-1.min-1, n = 6; or hydralazine, 16 +/- 2 mg, n = 4) while central venous pressure was held constant by simultaneous volume expansion. Changes in arterial pressures were compared with changes in heart rate and MSNA over 3-min periods of vasodilator infusion during which we observed increases in systolic and pulse pressures with simultaneous decreases in mean and diastolic pressures. During vasodilator infusion, there were increases in systolic (124.2 +/- 2.1 to 131.7 +/- 2.9 Torr, P less than 0.001) and pulse pressures (57.0 +/- 2.2 to 72.7 +/- 2.7 Torr, P less than 0.001) although mean arterial pressure fell (88.0 +/- 2.6 to 80.4 +/- 2.7 Torr, P less than 0.001) because of decreases in diastolic pressure (67.2 +/- 3.0 to 59.0 +/- 2.7 Torr, P less than 0.001). The changes in arterial pressures were accompanied by simultaneous increases in heart rate (66.4 +/- 3.0 to 92.6 +/- 4.8 beats/min, P less than 0.001) and MSNA (327 +/- 59 to 936 +/- 171 U, P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelin and ET(A) receptors in long-term arterial pressure homeostasis in conscious nonhuman primates

This study was designed to quantify the long-term contribution of endogenous endothelin-1 (ET-1) and ET(A) receptors to the regulation of arterial pressure under normal conditions in nonhuman primates. Therefore, mean arterial pressure (MAP) and heart rate were measured 24 h/day with the use of telemetry techniques in conscious cynomolgus monkeys under control conditions, during administration of an ET(A) selective receptor antagonist (ABT-627; 5 mg/kg, 2 times a day by mouth, 4 days), and a 6-day posttreatment period. Systemic ET(A) blockade reduced MAP (24 h) from 89 +/- 3 to 82 +/- 2 and 79 +/- 2 mmHg on days 1 and 4, respectively. Subsequently, MAP remained suppressed for 3 days posttreatment. Heart rate increased from 111 +/- 5 to 122 +/- 4 and 128 +/- 6 beats/min on days 1 and 4 of ABT-627, respectively, and remained above control for 3 days posttreatment. Plasma ET-1 concentration increased from 1.0 +/- 0.3 to 1.9 +/- 0.4 pg/ml in response to ABT-627 (day 4) but decreased to control values 4 days posttreatment. These data demonstrate a physiologically important role for endogenous ET-1 and ET(A) receptors in the long-term regulation of arterial pressure and plasma ET-1 levels in the conscious nonhuman primate.     

Effects of calcium binding and of EDTA and CaEDTA on the clotting of bovine fibrinogen by thrombin

Studies were carried out at pH 7.0 and gamma/2 0.15 before addition of CaCl2 or EDTA. Clotting time, tau, at 3.03 microM fibrinogen and 0.91 u/ml thrombin was determined for equilibrium systems. With added Ca2+, tau decreases, from tau 0 at 0 added Ca2+ (mean, 29.7 +/- 3 s), by approximately 3 s at 5 mM added Ca2+. With added EDTA, tau increases sigmoidally from tau 0 at 0 EDTA to a maximum (mean tau m = 142 +/- 23 s) at approximately 200 microM EDTA. tau then decreases slightly to a minimum at approximately 1.3 mM and finally increases to infinity at approximately 10 mM EDTA. Between 0 and 1.3 mM EDTA, effects on clotting time are completely reversed by adding Ca2+ and, after equilibration at 400 microM EDTA, tau is independent of EDTA concentration. Thus, up to 400 microM EDTA, effects on clotting time are attributed to decreasing fibrinogen bound Ca2+. Between 5 mM Ca2+ and 200 microM EDTA it is assumed that an equilibrium distribution of fibrinogen species having 3, 2, 1, or 0 bound calcium ions is established and that a clotting time is determined by the sum of products of species fractional abundance and pure species clotting time. Analysis indicates that pure species clotting times increase proportionately with decreasing Ca2+ binding, binding sites are nearly independent, and the microscopic association constant for the first bound Ca2+ is approximately 4.9 X 10(6) M-1. Effects of adding Ca2+ at times t1 after thrombin addition to systems initially equilibrated at 200 microM EDTA were determined. Analysis of the relation between tau and t1 indicates that as Ca2+ binding decreases, rate constants for release of B peptides decrease less than those for release of A peptides. As EDTA concentration is increased above 1.3 mM, inhibitory effects of EDTA and CaEDTA progressively increase.     

Prolactin and LH release in response to vasoactive intestinal peptide (VIP) administration in spontaneously hypertensive and normotensive rats.

Vasoactive intestinal peptide (VIP) was injected intravenously at a dose of 10 micrograms in spontaneously hypertensive and normotensive Wistar-Kyoto rats. In order to evaluate the hemodynamic and hormonal effects of this peptide, the mean arterial pressure, heart rate as well as a serum rLH and rPRL levels, the contents of LH-RH in hypothalamus and the content of LH in pituitary tissue were determined. The same procedure was applied in rats receiving placebo. Serum rPRL concentration was measured additionally after combined administration of VIP+dopamine. VIP injection produced a decrease in mean arterial pressure and an increase in heart rate in both spontaneously hypertensive and normotensive rats. Serum rPRL concentration was significantly increased at 10 minutes after injection. The combined therapy (VIP+dopamine) partially inhibited this response. Serum rLH concentration, the content of LH-RH in hypothalamic tissue as well as the content of pituitary LH after VIP injection in spontaneously hypertensive and normotensive rats did not differ from the values obtained for the control group. Conclusions: 1. VIP injection produced the dramatic hypotensive effects in hypertensive rats; 2. A marked increase in PRL concentration in response to VIP was partially inhibited by dopamine in hypertensive and normotensive rats; 3. VIP injection did not change LH-RH and LH release in both hypertensive and normotensive rats.