Functional correlates of central arterial geometric phenotypes.

We have assessed the functional correlates of common carotid artery (CCA) arterial geometry, derived by combining a measure of vascular mass (VM) with the wall-to-lumen (W/L) ratio in both untreated hypertensive (HT) and normotensive (NT; blood pressure <140/90 mm Hg) subjects of a broad age span (30 to 79 years) of both genders. Brachial systolic, diastolic, and pulse (SBP, DBP, PP) pressures; CCA SBP and PP; CCA diameter (D); intima-media thickness (IMT); relative distensibility; circumferential wall stress (MBPxW/L); fluid shear stress (FSS); strain; augmentation index (AGIh); and aortic pulse wave velocity (PWV) were measured in 680 NT and 635 untreated HT Taiwanese men and women. Carotid geometric phenotypes (CGPs) were derived from ultrasonographic measures of VM and W/L ratio. A normal CGP (CGP1) was defined as that within the 95th NT percentile of age- and gender-specific VM and W/L means. Three "deviant" CGPs were defined as follows: CGP2 or remodeling, ie, a normal VM coupled with an increased W/L; CGP3 or hypertrophy, ie, an increase in both VM and W/L; and CGP4 or hypertrophy with dilation, ie, an increased VM with normal W/L. The prevalence of specific CGPs in the total sample was 83.4% for CGP1, 5.5% for CGP2, 2.2% for CGP3, and 8.9% for CGP4. Compared with CGP1, all deviant CGPs had increased carotid resistance, had higher CCA circumferential wall stress, and varied in blood flow velocity. Compared with CGP1, CGP2 subjects were more likely to be women (69.3% versus 45.9%), were on average 10 years older, and had similar central and brachial BP levels, PWV, and AGIh but had increased strain, higher distensibility, lower flow, and a higher FSS. CGP3 subjects did not differ in age or gender but had a higher prevalence of HT; higher circumferential stress, PWV, and distensibility; and lower flow, as well as a trend toward higher SBP, PP, and AGIh and lower FSS. CGP4 subjects did not differ in age or gender but exhibited higher AGIh and aortic PWV, lower distensibility and FSS, and unchanged strain and flow. CGP4 was the only deviant CGP in which the average brachial or central arterial pressures were significantly increased. CGP4 subjects also had the highest prevalence of HT among all the CGPs (77.8% versus 45% in CGP1). CGPs exhibit some common mechanical or functional properties but each also exhibits a unique profile. Although differing quantitatively in NT and HT and at young and older age, the characteristic functional profile of a given CGP is preserved, regardless of age or BP status. A normal CGP is characterized by a low circumferential wall stress and high FSS. Each deviant CGP is characterized by a unique combination of increased circumferential wall stress, with variable FSS, strain, distensibility, central BP, and late pressure augmentation. The interplay among these factors, particularly circumferential wall and FSS, likely determines the CGP; conversely, the resultant CGP may modulate the FSS and wall stress for a given pressure and flow.

Measurement variation of aortic pulse wave velocity in the elderly.

Accurate and reproducible measures are required to study arterial stiffness in human populations. The reproducibility of aortic pulse wave velocity was evaluated in 14 participants from a population-based study of cardiovascular disease in the elderly. Three data files were collected per participant by each of two sonographers and files were read by two readers. Seven of the 14 participants returned for a second visit 1 week later to assess between-visit variability. Reproducibility was evaluated with Pearson and intraclass correlations and by the absolute value of the difference between replicate values. The overall reliability coefficient was rI = 0.77. Between-sonographer, between-reader, and between-visit correlations were rP = 0.80 to 0.87, rP = 0.73 to 0.89 and rP = 0.63. The mean absolute value of the difference between replicates was 59.4 to 94.0 cm/sec and 88.7 to 112.8 cm/sec for sonographers and readers, respectively. These results indicate that the mean PWV measure is reproducible even when sonographers and readers are newly trained.

High-density lipoprotein cholesterol and vascular stiffness at baseline in the activity counseling trial.

In a middle-aged patient population, age was associated with stiffer vessels and high-density lipoprotein cholesterol with more elastic vessels. High-density lipoprotein cholesterol may be an indirect indicator of aerobic capacity or of less atherosclerosis, suggesting mechanisms for preserving vascular integrity.

Cardiac synthesis, processing, and coronary release of enkephalin-related peptides.

Although preproenkephalin mRNA is abundant in the heart, the myocardial synthesis and processing of proenkephalin is largely undefined. Isolated working rat hearts were perfused to determine the rate of myocardial proenkephalin synthesis, its processing into enkephalin-containing peptides, their subsequent release into the coronary arteries, and the influence of prior sympathectomy. Enkephalin-containing peptides were separated by gel filtration and quantified with antisera for specific COOH-terminal sequences. Proenkephalin, peptide B, and [Met(5)]enkephalin-Arg(6)-Phe(7) (MEAP) comprised 95% of the extracted myocardial enkephalins (35 pmol/g). Newly synthesized enkephalins, estimated during a 1-h perfusion with [(14)C]phenylalanine (4 pmol x h(-1) x g wet wt(-1)), were rapidly cleared from the heart during a second isotope-free hour. Despite a steady release of enkephalins into the coronary effluent (4 pmol x h(-1) x g wet wt(-1)), enkephalin replacement apparently exceeded its release, and tissue enkephalins actually accumulated during hour 2. In contrast to the tissue, methionine-enkephalin accounted for more than half of the released enkephalin. Chemical sympathectomy produced an increase in total enkephalin content similar to that observed after 2-h control perfusion. This observation suggested that the normal turnover of myocardial enkephalin may depend in part on continued sympathetic influences.

beta2-adrenergic cAMP signaling is uncoupled from phosphorylation of cytoplasmic proteins in canine heart.

BACKGROUND: Recent studies of beta-adrenergic receptor (beta-AR) subtype signaling in in vitro preparations have raised doubts as to whether the cAMP/protein kinase A (PKA) signaling is activated in the same manner in response to beta2-AR versus beta1-AR stimulation. METHODS AND RESULTS: The present study compared, in the intact dog, the magnitude and characteristics of chronotropic, inotropic, and lusitropic effects of cAMP accumulation, PKA activation, and PKA-dependent phosphorylation of key effector proteins in response to beta-AR subtype stimulation. In addition, many of these parameters and L-type Ca2+ current (ICa) were also measured in single canine ventricular myocytes. The results indicate that although the cAMP/PKA-dependent phosphorylation cascade activated by beta1-AR stimulation could explain the resultant modulation of cardiac function, substantial beta2-AR-mediated chronotropic, inotropic, and lusitropic responses occurred in the absence of PKA activation and phosphorylation of nonsarcolemmal proteins, including phospholamban, troponin I, C protein, and glycogen phosphorylase kinase. However, in single canine myocytes, we found that beta2-AR-stimulated increases in both ICa and contraction were abolished by PKA inhibition. Thus, the beta2-AR-directed cAMP/PKA signaling modulates sarcolemmal L-type Ca2+ channels but does not regulate PKA-dependent phosphorylation of cytoplasmic proteins. CONCLUSIONS: These results indicate that the dissociation of beta2-AR signaling from cAMP regulatory systems is only apparent and that beta2-AR-stimulated cAMP/PKA signaling is uncoupled from phosphorylation of nonsarcolemmal regulatory proteins involved in excitation-contraction coupling.

Opposing effects of alpha 1-adrenergic receptor subtypes on Ca2+ and pH homeostasis in rat cardiac myocytes.

We examined the effect of alpha 1-adrenergic receptor (AR) subtypes on contraction, cytosolic Ca2+ concentration ([Ca2+]i), and cytosolic pH (pHi) of rat ventricular myocytes loaded with the Ca2+ indicator indo 1 or the pH indicator carboxyseminaphthorhodafluor-1. Nonselective alpha 1-AR stimulation was effected with phenylephrine plus nadolol. alpha 1-AR subtype stimulation was achieved with alpha 1-AR and chloroethylclonidine (CEC) or with alpha 1-AR and WB-4101. Cells were in bicarbonate buffer with 0.5 mM Ca2+ and were electrically stimulated at 0.5 Hz. Results show that 1) nonselective alpha 1-AR stimulation increased twitch and [Ca2+]i transient amplitudes, myofilament response to Ca2+, and pHi; 2) alpha 1-AR plus CEC increased twitch and [Ca2+]i transient amplitudes and also enhanced myofilament response to Ca2+ via cytosolic alkalinization; 3) alpha 1-AR plus WB-4101 decreased twitch and [Ca2+]i transient amplitudes and also pHi; and 4) cytosolic acidification due to alpha 1-AR plus WB-4101 was abolished by protein kinase C inhibition (staurosporine pretreatment) or downregulation (prolonged exposure to phorbol esters). In summary, the net effects of alpha 1-adrenergic stimulation on contraction, [Ca2+]i, and pHi are due to opposing WB-4101- and CEC-sensitive alpha 1-AR subtype signaling pathways.

Modulation of the transient outward current in adult rat ventricular myocytes by polyunsaturated fatty acids.

With the whole cell patch-clamp technique, we studied the effects of the n-3 and n-6 polyunsaturated fatty acids (PUFAs), linoleic (C18:2n-6), eicosapentaenoic (C20:4n-3), docosahexaenoic (C22:5n-3), and arachidonic (AA; C20:4n-6) acids, on K+ currents in rat ventricular myocytes. At low concentrations (5-10 microM) all PUFAs except AA inhibited, by approximately 40%, the transient outward current (I(to)) without affecting other K+ currents and markedly prolonged the action potential (AP). AA inhibited I(to) but also augmented a sustained depolarization-induced outward K+ current (Isus); the latter effect did not occur in the presence of 4-aminopyridine or with eicosatetraynoic acid, a nonmetabolizable analog of AA. Higher concentrations of PUFAs (20-50 microM) further inhibited I(to) and also inhibited Isus. Thus, at high concentrations, PUFAs have a nonspecific effect on several K+ channels; at low concentrations, PUFAs preferentially inhibit I(to) and prolong the AP.

Opioid peptide receptor stimulation reverses beta-adrenergic effects in rat heart cells.

Opioid peptide receptor (OPR) agonists are co-released with the beta-adrenergic receptor (beta-AR) agonist norepinephrine (NE) from nerve terminals in the heart during sympathetic stimulation. Whereas recent studies indicate that OPR and beta-AR coexist on the surface of cardiac myocytes, whether significant "cross talk" occurs between OPR and beta-AR signaling cascades within heart cells is unknown. In the present study we demonstrate a marked effect of delta-OPR stimulation to modulate beta-adrenergic responses in single isolated rat ventricular myocytes. Nanomolar concentrations (10(-8) M) of the OPR agonist leucine enkephalin (LE), a naturally occurring delta-opioid peptide, inhibited NE-induced increases in sarcolemmal L-type Ca2+ current, cytosolic Ca2+ transient, and contraction. The antiadrenergic effect of LE was pertussis toxin sensitive and abolished by naloxone, an opioid receptor antagonist. In contrast, LE was unable to inhibit the positive inotropic effects induced by equipotent concentrations of 8-(4 chlorophenylthio)-adenosine 3',5'-cyclic monophosphate, a cell-permeant adenosine 3',5'-cyclic monophosphate analog, or by the non-receptor-induced increase in contraction by elevated bathing Ca2+ concentration. These results indicate that an interaction of the OPR and beta-AR systems occurs proximal to activation of the adenosine 3',5'-cyclic monophosphate-dependent protein kinase of the beta-AR intracellular signaling pathway. This modulation of beta-adrenergic effects by OPR activation at the myocyte level may have important implications in the regulation of cardiac Ca2+ metabolism and contractility, particularly during the myocardial response to stress.

Initial contractile response of isolated rat heart cells to halothane, enflurane, and isoflurane.

BACKGROUND: In several beating cardiac muscle preparations, a short-lived increase in twitch tension or amplitude has been observed when they were exposed abruptly to solutions containing halothane or enflurane. As exposure to the anesthetics was continued, the expected negative inotropic effect became evident after the short-lived increase in twitch. No such increase in twitch has been reported during exposure to isoflurane. It has been hypothesized that this short-lived increase in twitch is caused by an enhancement of calcium release from the sarcoplasmic reticulum, but other mechanisms have not been excluded. METHODS: Freshly isolated, single rat ventricular cells were stimulated to beat at room temperature and abruptly exposed to solutions containing halothane (0.25-0.64 mM), enflurane (0.69-1 mM), or isoflurane (0.31-0.54 mM). During these exposures, twitch amplitude was measured and intracellular calcium concentration was followed using the calcium-sensitive dye indo-1. In some experiments, the whole-cell patch-clamp technique was used to measure membrane current. In addition, in several cells the sarcoplasmic reticulum calcium content was assessed through the response to brief pulses of caffeine. RESULTS: Both the twitch amplitude and the intracellular calcium transient were increased temporarily in cells abruptly exposed to halothane or enflurane. No such behavior was found with isoflurane. After continued exposure to all three agents, both the twitch amplitude and the calcium transient were less than control. During the beats exhibiting an increase in twitch, no alteration in the relation between cell length (twitch amplitude) and the intracellular calcium transient was found compared with control conditions. In addition, the temporary increase in twitch amplitude occurred in cells contracting under voltage-clamp control when halothane was introduced, and it was not associated with any increase in the calcium current. The sarcoplasmic reticulum calcium content at the time of the halothane-induced increase in twitch also was not increased. CONCLUSIONS: The short-lived increase in twitch after abrupt exposure to halothane or enflurane is related to increased intracellular calcium during the beat and not to any changes in myofilament sensitivity to calcium. Because these results eliminate most alternative explanations for this phenomenon, the authors conclude that halothane, and probably also enflurane, increases the fraction of calcium released from the sarcoplasmic reticulum with each heart beat. Isoflurane appears to lack this action.

Actomyosin interaction modulates resting length of unstimulated cardiac ventricular cells.

We sought to determine whether resting or diastolic cardiac myocyte length during low stimulation rates is regulated by myofilament interaction. Cytosolic Ca2+ concentration ([Ca2+]i, via indo 1 fluorescence) and length, in the presence and absence of 2,3-butanedione monoxime (BDM), a potent inhibitor of force production in striated muscle, were measured in rat and guinea pig cardiac myocytes at rest and after electrical stimulation. In tetanized cells BDM reduced steady contraction amplitudes for a given [Ca2+]i. In an actomyosin-sliding filament assay without Ca2+ or regulatory proteins, BDM decreased actin filament velocity along myosin. BDM increased both diastolic and resting cell lengths without changes in [Ca2+]i. The resting cell length also increased when [Ca2+]i was reduced by removing extracellular Ca2+, an effect further enhanced by BDM and by loading cells with the intracellular Ca2+ chelator, 1,2-bis(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethylester. Thus myofilament interaction is present in cardiac cells, both at rest or during low rates of stimulation, and this myofilament interaction is regulated, in part, by the ambient [Ca2+]i.

L- and T-type calcium currents differ in finch and rat ventricular cardiomyocytes.

We compared L-type Ca current (ICaL) and T-type Ca current (ICaT) in finch and rat myocytes, using whole-cell patch clamp techniques. Cell capacitance averaged 50 +/- 4 pF in finch (n = 25) v 145 +/- 8 pF in rat (n = 38) cells, P < 0.001. In cells bathed in 1 mM Cao at 22 degrees C, peak ICaL amplitude, during a voltage clamp step (10 mM EGTA in pipette) from -45 mV to -5 mV, averaged 10.5 +/- 0.3 pA/pF in finch v 6.9 +/- 0.6 pA/pF, P < 0.001 in rat cells. ICaL inactivation kinetics were faster in finch (4.6 +/- 0.3 ms) than in rat (13.4 +/- 1.3 ms) cells. P < 0.001. ICaT was not detectable in rat cells (2 mM bathing [Ca]); but in finch cells, a large ICaT which averaged 6.8 +/- 1.4 pA/pF was activated at -30 mV and was relatively insensitive to nitrendipine (0.1 microM). The distinctive features of ICaL and ICaT in finch cells may have a role in the ability of the finch to achieve a very rapid heart rate. They may also facilitate excitation-Ca2+ release coupling in finch ventricular cells which are devoid of T tubules and have relatively few junctions between the sarcolemma and the sarcoplasmic reticulum.

Endoplasmic reticulum Ca2+ depletion unmasks a caffeine-induced Ca2+ influx in human aortic endothelial cells.

Intracellular Ca2+ pools contribute to changes in cytosolic [Ca2+] ([Ca2+]i), which play an important role in endothelial cell signaling. Recently, endothelial ryanodine-sensitive Ca2+ stores were shown to regulate agonist-sensitive intracellular Ca2+ pools. Since caffeine binds the ryanodine Ca2+ release channel on the endoplasmic reticulum in a variety of cell types, we examined the effect of caffeine on [Ca2+]i in human aortic endothelial cell monolayers loaded with the fluorescent probe indo 1. Under baseline conditions, 10 mmol/L caffeine induced a small increase in [Ca2+]i from 86 +/- 10 to 115 +/- 17 nmol/L (mean +/- SEM); this effect was similar to that of 5 mumol/L ryanodine and was unaffected by buffer Ca2+ removal. After depletion of an intracellular Ca2+ store by the irreversible endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin (1 mumol/L), ryanodine did not affect [Ca2+]i. In contrast, caffeine induced a large rapid increase in [Ca2+]i (176 +/- 19 to 338 +/- 35 nmol/L, P < .001) after thapsigargin exposure; this effect of caffeine was only observed when extracellular Ca2+ was present. A similar increase in [Ca2+]i was induced by caffeine after depletion of ryanodine- and histamine-sensitive Ca2+ stores or after pretreatment with the endoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid (10 mumol/L). Thus, under baseline conditions the effect of caffeine on [Ca2+]i is similar to that of ryanodine and appears to be due to the release of an intracellular store. However, after depletion of an endoplasmic reticulum Ca2+ store, caffeine, but not ryanodine, stimulates Ca2+ influx, resulting in a large increase in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced threshold for myocardial cell calcium intolerance in the rat heart with aging.

To determine whether advancing age is accompanied by a reduced Ca2+ tolerance, we measured Ca(2+)-dependent diastolic pressure, prolonged relaxation and systolic functional deterioration, spontaneous sarcoplasmic reticulum (SR)-generated Ca2+ oscillations [detected as scattered laser light intensity fluctuations (SLIF)], aftercontractions, and ventricular fibrillation in isolated, isovolumic, atrioventricular-blocked intact hearts from 24- to 26-mo (old) and 6- to 8-mo (young) male Wistar rats. In enzymatically isolated single cardiac myocytes, the likelihood of the occurrence of spontaneous contractile waves driven by spontaneous SR Ca2+ release was also determined. In response to stepwise increase in perfusate Ca2+ concentration (Cao), a reduction in the maximum developed pressure accompanied by an elevation in end-diastolic pressure and a prolonged contraction duration was observed at lower Cao in old vs. young hearts (P < 0.01 for each parameter). Furthermore, Ca(2+)-dependent ventricular fibrillation occurred during pacing in six old but in no young hearts (P < 0.01), aftercontractions were observed in seven old vs. one young heart (P < 0.01), and SLIF increased to a greater extent in old vs. young hearts. In single cardiac myocytes, spontaneous contractile waves occurred more frequently with increasing age (P < 0.01). These results indicate that aging is associated with an increased likelihood for the occurrence of SR-generated Ca2+ oscillations and functional abnormalities that result from these oscillations.

cGMP prevents delayed relaxation at reoxygenation after brief hypoxia in isolated cardiac myocytes.

Previous studies in isolated cardiac myocytes suggest that impaired relaxation during reoxygenation after brief hypoxia results from abnormal Ca(2+)-myofilament interaction. Recent studies indicate that guanosine 3',5'-cyclic monophosphate (cGMP)-elevating interventions selectively enhance myocardial relaxation. We investigated the effect of 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) on posthypoxic relaxation in single rat myocytes, with simultaneous measurement of contraction and intracellular Ca2+ (indo 1 fluorescence). In control myocytes (n = 11), reoxygenation after 10 min of hypoxia markedly prolonged time to peak shortening (+36.5 +/- 4.2%) and half-relaxation time (+75.7 +/- 11.3% cf. normoxic values; both P < 0.001) and reduced diastolic length but did not change cytosolic Ca2+. Under normoxic conditions, 50 microM 8-BrcGMP slightly reduced time to peak shortening and half-relaxation time and increased diastolic length but did not alter cytosolic Ca2+. In the presence of 8-BrcGMP, there was no posthypoxic delay in twitch relaxation nor was there a decrease in diastolic length (half-relaxation time -5.8 +/- 3.3% cf. normoxic values; P < 0.05 cf. control group; n = 11). Cytosolic Ca2+ remained unaltered. Thus, 8-BrcGMP fully prevents impaired posthypoxic relaxation in isolated cardiac myocytes, probably by altering Ca(2+)-myofilament interaction.

Effects of sarcoplasmic reticulum Ca2+ load on the gain function of Ca2+ release by Ca2+ current in cardiac cells.

We studied the effects of variable sarcoplasmic reticulum (SR) Ca2+ loading on changes in the gain index of Ca2+ release from the SR, measured as the ratio of the amount of Ca2+ released to the magnitude of the Ca2+ current (ICa) integrated for the initial 20 ms of the depolarization, in whole cell voltage-clamped rat ventricular myocytes dialyzed with the Ca2+ indicator indo 1 salt at 23 degrees C. Changes in ICa were measured directly, and changes in the SR Ca2+ release were indexed by changes in the amplitudes and rates of rise of cytosolic Ca2+ (Ca2+i) transients. The SR Ca2+ load was graded by the duration of conditioning voltage-clamp steps and verified by caffeine-dependent Ca2+i transients. A train of abbreviated (from 100 to 20 ms) voltage-clamp depolarizations, which triggers SR Ca2+ release but fails to replenish the SR with Ca2+, diminished the SR Ca2+ load by 56 +/- 5%, did not alter peak ICa but reduced the amplitudes of the ICa-dependent Ca2+i transients by 52 +/- 3%, and decreased the gain index by 60 +/- 3% (SE; n = 5 or 6). Changes in the amplitudes of Ca2+i transients elicited by ICa and changes in the gain index were linearly correlated (r2 = 0.83 and 0.79, respectively; P < 0.001 for each) with changes in amplitudes of Ca2+i transients elicited by caffeine pulses applied in lieu of the respective voltage-clamp pulses.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-associated changes in beta-adrenergic modulation on rat cardiac excitation-contraction coupling.

Previous studies have demonstrated that the ability of beta-adrenergic receptor (beta AR) stimulation to increase cardiac contractility declines with aging. In the present study, the control mechanisms of excitation-contraction (EC) coupling, including calcium current (ICa), cytosolic Ca2+ (Cai2+) transient and contraction in response to beta AR stimulation were investigated in ventricular myocytes isolated from rat hearts of a broad age range (2, 6-8, and 24 mo). While the baseline contractile performance and the Cai2+ transient did not differ markedly among cells from hearts of all age groups, the responses of the Cai2+ transient and contraction to beta-adrenergic stimulation by norepinephrine (NE) diminished with aging: the threshold concentration and the ED50 increased in rank order with aging; the maximum responses of contraction and Cai2+ transient decreased with aging. Furthermore, the efficacy of beta AR stimulation to increase ICa was significantly reduced with aging, and the diminished responses of the contraction and Cai2+ transient amplitudes to NE were proportional to the reductions in the ICa response. These findings suggest that the observed age-associated reduction in beta AR modulation of the cardiac contraction is, in part at least, due to a deficit in modulation of Cai2+, particularly the activity of L-type calcium channels.

8-bromo-cGMP reduces the myofilament response to Ca2+ in intact cardiac myocytes.

The role of cGMP in myocardial contraction is not established. Recent reports suggest that nitric oxide, released by endothelial cells or within myocytes, modifies myocardial contraction by raising cGMP. We studied the effects of 8-bromo-cGMP (8bcGMP, 50 mumol/L) on contraction (cell shortening) and simultaneous intracellular Ca2+ transients (indo 1 fluorescence ratio) in intact adult rat ventricular myocytes (0.5 Hz and 25 degrees C) 8bcGMP reduced myocyte twitch amplitude and time to peak shortening (-19.6 +/- 4.2% and -17.6 +/- 1.3%, respectively) and increased steady-state diastolic cell length (+0.6 +/- 0.1 microns, mean +/- SEM, n = 8; all P < .05) but had no effect on shortening velocity, systolic or diastolic fluorescence ratio, or time to peak fluorescence ratio (all P = NS). In 7 of 13 myocytes, this negative inotropic effect was preceded by a transient positive inotropic effect, with small increases in twitch amplitude, shortening velocity, and cytosolic Ca2+ transient. Analysis of 8bcGMP effects on both the dynamic and steady-state relation between cell shortening and intracellular Ca2+ (during twitch contraction and tetanic contraction, respectively) indicated reduction in the myofilament response to Ca2+ in all cases. These 8bcGMP effects were inhibited by KT5823 (1 mumol/L), an inhibitor of cGMP-dependent protein kinase, or by the presence of isoproterenol (3 nmol/L). 8bcGMP had no effect on cytosolic pH in cells (n = 4) loaded with the fluorescent probe carboxyseminaphthorhodafluor-1. These data indicate that cGMP may modulate myocardial relaxation and diastolic tone by reducing the relative myofilament response to Ca2+, probably via cGMP-dependent protein kinase.

A functional ryanodine-sensitive intracellular Ca2+ store is present in vascular endothelial cells.

The presence of the ryanodine receptor was recently demonstrated in vascular and endocardial endothelium, but its function has not been established. We investigated whether functional ryanodine-sensitive Ca2+ stores are present in cultured endothelial cells from rat aorta (RAECs), human aorta (HAECs), human umbilical vein (HUVECs), and bovine pulmonary artery (BPAECs) and what role these may play in intracellular Ca2+ regulation. Under resting conditions, HAECs, BPAECs, and HUVECs demonstrated a slow increase in intracellular Ca2+ (indexed by indo 1 fluorescence) on exposure to 5 mumol/L ryanodine, whereas RAECs did not. However, after an initial bradykinin exposure in RAECs, ryanodine markedly blunted the rapid increase in Ca2+ on a second exposure to bradykinin. In HUVECs, ryanodine in buffer with 1.5 mmol/L Ca2+ did not inhibit the agonist-sensitive Ca2+ increase, whereas it blunted the rapid increase in Ca2+ on histamine exposure in buffer with 5 mmol/L Ca2+, suggesting that increasing [Ca2+] enhances the binding of ryanodine to its receptor. Thus, functional ryanodine-sensitive Ca2+ stores are present in vascular endothelial cells. These appear to be involved in regulation of Ca2+ storage and release from agonist-sensitive intracellular compartments.

Stereoselective actions of thiadiazinones on canine cardiac myocytes and myofilaments.

Thiadiazinones are cardiotonic agents that have potent, direct, and stereoselective actions on the myofilament response to Ca2+ in intact myocardium. Their mechanism of action is unknown. We studied the effects of racemic thiadiazinone, EMD 53998 (5-[1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydro-6-quinolyl]-6-meth yl-3,6- dihydro-2H-1,3,4-thiadiazin-2-one), and its enantiomers on Ca2+ signaling in myocytes, myofilaments, and myofilament proteins. Intact canine ventricular myocytes responded to the positive enantiomer, EMD 57033, with an increase in the extent of shortening during twitch contractions without increasing the peak amplitude of the Ca2+ transient. The negative enantiomer, EMD 57439, also increased the extent of shortening, but in this case there was a concentration-dependent increase in the peak amplitude of the Ca2+ transient. This is predicted from in vitro data showing that this enantiomer is a relatively potent inhibitor of phosphodiesterase activity. There was no effect of EMD 57439 on the relation between pCa and actomyosin Mg-ATPase activity of canine heart myofibrils. In contrast, EMD 57033 shifted the pCa-Mg-ATPase activity relation to the left. There was no effect of either enantiomer on Ca2+ binding to myofilament troponin C. Moreover EMD 57033, but not EMD 57439, stimulated actomyosin ATPase activity of myofilament preparations in which either troponin or troponin-tropomyosin had been extracted. EMD 57033 had no effect on Mg-ATPase activity of pure ventricular myosin. EMD 57033 also stimulated the velocity of actin filament sliding on myosin heads adhered to nitrocellulose-coated glass coverslips. We propose that the action of EMD 57033 is at the actin-myosin interface on a "receptor" that may be on actin or the crossbridge. Drug binding to this domain appears to reverse the inhibition of actin-myosin interactions by troponin-tropomyosin and also to promote transition of crossbridges from weak to strong force-generating states.

Pathological characterization of male Wistar rats from the gerontology research center.

Male Wistar rats aged 6-26 months were obtained from the colony of The Gerontology Research Center of the National Institute on Aging, and pathological profiles were assessed. One hundred animals were sacrificed at 6, 12, 18, 21, 24, and 26 months and used for cross-sectional determinations; also, 150 animals were followed longitudinally and sacrificed when clinical signs of moribundity appeared. Renal disease contributed the most common pathology observed in both studies (found in over 70% of the animals examined), with neoplasms a secondary problem (pituitary tumors were by far the most prevalent, with adenomas present in approximately 20% of the animals). This analysis represents the first complete pathological characterization of this commonly used rat model for aging research, and offers an opportunity for comparison with other rat strains.