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Biomedical subjects

R A Kelly

Publications and source records attributed to R A Kelly.

At least 91 records · Page 5Linked to original sources
PubMed1994

Continual electric field stimulation preserves contractile function of adult ventricular myocytes in primary culture.

To model with greater fidelity the electromechanical function of freshly isolated heart muscle cells in primary culture, we describe a technique for the continual electrical stimulation of adult myocytes at physiological frequencies for several days. A reusable plastic cover was constructed to fit standard, disposable 175-cm2 tissue culture flasks and to hold parallel graphite electrodes along the long axis of each flask, which treated a uniform electric field that resulted in a capture efficiency of ventricular myocytes of 75-80%. Computer-controlled amplifiers were designed to be capable of driving a number of flasks concurrently, each containing up to 4 x 10(6) myocytes, over a range of stimulation frequencies (from 0.1 to 7.0 Hz) with reversal of electrode polarity after each stimulus to prevent the development of pH gradients around each electrode. Unlike quiescent, unstimulated myocytes, the amplitude of contraction, and velocities of shortening and relaxation did not change in myocytes paced at 3-5 Hz for up to 72 h. The maintenance of normal contractile function in paced myocytes required mechanical contraction per se, since paced myocytes that remained quiescent due to the inclusion of 2.5 microM verapamil in the culture medium for 48 h also exhibited a decline in contractility when paced after verapamil removal. Similarly, pacing increased peak calcium current compared with quiescent cells that had not been paced. Thus myocyte contraction at physiological frequencies induced by continual uniform electric field stimulation in short-term primary culture in defining medium maintains some biophysical parameters of myocyte phenotype that are similar to those observed in freshly isolated adult ventricular myocytes.

Animals↗
PubMed1993

Control of cardiac muscle cell function by an endogenous nitric oxide signaling system.

Nitric oxide (NO) synthesized from L-arginine is a ubiquitous intracellular chemical messenger and is involved in signal transduction in diverse mammalian cells, including vascular endothelium and neuronal tissues. The role of the NO-signaling pathway in the direct modulation of cardiac function is less well characterized. In this report, the effects of inhibitors of NO synthase (NOS) were examined in isolated neonatal and adult rat ventricular myocytes exposed to either muscarinic or adrenergic agonists. Carbachol (10 microM) caused a 91% inhibition of the spontaneous beating rate of cultured neonatal rat cardiac myocytes. N omega-monomethyl-L-arginine, an L-arginine analog that inhibits NOS, and methylene blue, an inhibitor of NO, blocked the negative chronotropic effect of carbachol but had no effect on the basal beating rate of these cells. The inhibition by N omega-monomethyl-L-arginine of the negative chronotropic effect of carbachol was reversed by adding excess L-arginine. The negative chronotropic effect of carbachol was also mimicked by analogs of cGMP, a second messenger implicated in mediating the action of NO in other cell types. Production of NO could be detected directly in carbachol-stimulated neonatal myocytes by using a reporter cell bioassay. The regulation of adrenergic responsiveness by the NO signaling system was also documented in studies of adult cardiac myocyte contractility. The NOS inhibitor N omega-nitro-L-arginine significantly increased the inotropic effect of the beta-adrenergic agonist isoproterenol on electrically stimulated adult rat ventricular myocytes, whereas this inhibitor had no effect on basal contractility. Inhibition of NO production by N omega-monomethyl-L-arginine in these cells, as measured by reporter cell bioassay, was also reversible with excess L-arginine. Thus, the physiologic response of isolated neonatal and adult ventricular myocytes to both muscarinic cholinergic and beta-adrenergic stimulation is mediated, at least in part, by products of an endogenous NOS.

Aging↗
PubMed1993

Subcutaneous injection of triamcinolone acetonide in the treatment of chronic vulvar pruritus.

OBJECTIVES: We studied the efficacy of subcutaneous corticosteroid injection as therapy for chronic vulvar pruritus. STUDY DESIGN: Adult women with symptoms of vulvar pruritus > 6 months were studied. After appropriate biopsies and cultures were performed, patients were included in the study group if they experienced limited relief with topical triamcinolone cream. Triamcinolone acetonide (15 to 20 mg) was injected subcutaneously and massaged into the affected vulvar tissue. RESULTS: Seventy-eight percent of patients (35/45) experienced relief of vulvar pruritus for > 1 month (mean duration 5.8 months). CONCLUSIONS: We found that subcutaneous injection triamcinolone acetonide provided relief of symptoms of chronic vulvar pruritus.

Adult↗
PubMed1993

Digoxin in heart failure: implications of recent trials.

Although supported by 2 centuries of anecdotal clinical evidence, the safety and efficacy of the cardiac glycosides for the treatment of congestive heart failure due to systolic ventricular dysfunction had never been rigorously examined by prospective clinical trials until the past decade. A reevaluation of the appropriate role of these drugs in modern cardiovascular pharmacology was prompted by the introduction in the 1970s of new classes of drugs for the treatment of congestive heart failure and supraventricular arrhythmias. Concurrently, several reports appeared, questioning the routine prescription of digoxin for the treatment of heart failure, particularly in patients in sinus rhythm. The majority of clinical trials published since 1980, most of which examined patients with New York Heart Association class II and III congestive heart failure, indicate that digoxin with or without concomitant administration of a vasodilator lessens symptoms and reduces the morbidity associated with congestive heart failure, particularly in patients with more advanced symptoms and ventricular dysfunction. The data on efficacy are less clear in support of the routine prescription of digoxin in the treatment of mild (class I and II) congestive heart failure. Although most recent trials attest to the relative safety and efficacy of digoxin in patients with congestive heart failure whose serum levels are maintained between 1 and 2 ng/ml, there is no conclusive evidence as yet that cardiac glycosides improve survival, as has been documented for vasodilators and, in particular, angiotensin-converting enzyme inhibitors. The National Institutes of Health-sponsored Digitalis Investigators Group (DIG) trial now underway should provide an answer to this question within the next few years.

Clinical Trials as Topic↗
PubMed1993

Isolation and characterization of human and rat cardiac microvascular endothelial cells.

Although reciprocal intercellular signaling may occur between endocardial or microvascular endothelium and cardiac myocytes, suitable in vitro models have not been well characterized. In this report, we describe the isolation and primary culture of cardiac microvascular endothelial cells (CMEC) from both adult rat and human ventricular tissue. Differential uptake of fluorescently labeled acetylated low-density lipoprotein (Ac-LDL) indicated that primary isolates of rat CMEC were quite homogeneous, unlike primary isolates of human ventricular tissue, which required cell sorting based on Ac-LDL uptake to create endothelial cell-enriched primary cultures. The endothelial phenotype of both primary isolates and postsort subcultured CMEC and their microvascular origin were determined by characteristic histochemical staining for a number of endothelial cell-specific markers, by the absence of cells with fibroblast or pericyte-specific cell surface antigens, and by rapid tube formation on purified basement membrane preparations. Importantly, [3H]-thymidine uptake was increased 2.3-fold in subconfluent rat microvascular endothelial cells 3 days after coculture with adult rat ventricular myocytes because of release of an endothelial cell mitogen(s) into the extracellular matrix, resulting in a 68% increase in cell number compared with CMEC in monoculture. Thus biologically relevant cell-to-cell interactions can be modeled with this in vitro system.

Animals↗
PubMed1993

Role of cation gradients in hypercontracture of myocytes during simulated ischemia and reperfusion.

We examined the relationship between transsarcolemmal cation gradients and hypercontracture of cardiac myocytes in ischemia and reperfusion using adult rat ventricular myocytes superfused with buffer mimicking normal or ischemic extracellular fluid. Contractile performance of electrically stimulated cells was recorded by an optical video system simultaneously with measurements of intracellular Ca2+ concentration ([Ca2+]i) using fura-2 or intracellular pH (pHi) using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. While cells were exposed to simulated ischemia buffer, the transsarcolemmal H+ gradient was abolished, [Ca2+]i transient stopped, and twitch contraction of myocytes ceased. Upon reperfusion with normal buffer, H+ gradient was quickly restored, Ca2+ transients restarted with transient increase in systolic Ca2+, and twitch contraction restarted with development of hypercontracture, which continued after [Ca2+]i returned to preischemic level even in the presence of near-normal concentrations of high-energy phosphates. When the transsarcolemmal proton, Na+, and Ca2+ gradients were altered so that Na+ entry via Na(+)-H+ exchange and Ca2+ entry via Ca(2+)-Na+ exchange were made less favorable, the transient systolic overshoot of Ca2+ at reperfusion and development of hypercontracture was largely avoided. These results suggest that Na+ and then Ca2+ entry via the Na(+)-H+ and Na(+)-Ca2+ exchangers, respectively, probably contribute to the increase in [Ca2+]i and hypercontracture of myocytes at time of reperfusion in this model.

Acidosis↗
PubMed1993

Adult rat ventricular myocytes cultured in defined medium: phenotype and electromechanical function.

We studied primary short-term cultures of adult rat ventricular myocytes in defined medium to determine whether phenotype and electromechanical function are maintained in rod-shaped, quiescent cells. Although > 80% of the myocytes retained their rod-shaped in vivo morphology for up to 72 h, contractile function as measured by cell edge motion declined 30-50% from 6 to 24 h, paralleling a 68% shortening of action potential duration. From 24 to 72 h, contractility remained unchanged. Ca2+ channel current density increased 55% after 24-48 h and then returned to the level of freshly isolated cells (9 +/- 1 pA/pF, mean +/- SE). Resting membrane potential (-71 +/- 1 mV) and action potential overshoot (34 +/- 3 mV) did not change. The ratio of alpha- to beta-myosin heavy chain mRNA and the level of cardiac alpha-actin mRNA were maintained for 8 days. Thus quiescent adult rat ventricular myocytes in defined medium undergo extensive phenotypic adaptation within 72 h of isolation, despite maintenance of a rod-shaped morphology and stable levels of contractile protein mRNA, which may limit their suitability for electrophysiological and contractile function studies.

Animals↗
PubMed1993

Cell-cell signaling between adult rat ventricular myocytes and cardiac microvascular endothelial cells in heterotypic primary culture.

It is unclear whether signaling between endothelial cells and muscle cells within ventricular myocardium, known to be important during cardiac development, remains physiologically relevant in the adult heart. Also, the mechanisms regulating the synthesis and activation of locally acting autacoids such as endothelins, cytokines known to have potent effects on contractile function and gene expression in cardiac myocytes, are unknown, as their cells of origin within ventricular muscle. Microvascular endothelial cells isolated from ventricular tissue of adult rats do not express endothelins constitutively. However, the appearance of preproendothelin mRNA can be increased in cardiac microvascular endothelial cells by heterotypic primary culture with adult rat ventricular myocytes. Cell-cell contact, or at least close apposition, appears to be necessary to increase preproendothelin mRNA, as medium conditioned by ventricular myocytes alone was ineffective when applied to monocultures of microvascular endothelial cells. The level of TGF beta precursor mRNA is also markedly increased in microvascular endothelial cells in coculture and precedes the appearance of endothelin precursor transcripts. In coculture, TGF beta acts as an autocrine cytokine, increasing endothelin precursor mRNA and inhibiting the rate of microvascular endothelial cell proliferation. This regulation of endothelial cell phenotype in heterotypic primary cultures suggests that dynamic, reciprocal cell-cell signaling may also be occurring between microvascular endothelium and ventricular myocytes in vivo.

Amino Acid Sequence↗
PubMed1993

Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium.

The mechanism by which soluble mediators of immune cell origin depress myocardial contractility, either globally as in systemic sepsis, or regionally in areas of inflammatory myocardial infiltrates, remains unclear. When freshly isolated ventricular myocytes from adult rat hearts were preincubated for at least 24 h in medium conditioned by endotoxin (LPS)-activated rat alveolar macrophages, their subsequent inotropic response to the beta-adrenergic agonist isoproterenol was reduced from 225 +/- 19% to 155 +/- 10% of the baseline amplitude of shortening (mean +/- SEM, P < 0.05). Neither baseline contractile function nor the contractile response to high extracellular calcium were affected. To determine whether an endogenous nitric-oxide (NO)-signaling pathway within ventricular myocytes was responsible for their decreased responsiveness to isoproterenol, the L-arginine analogue L-NMMA was added to the preincubation medium. While L-NMMA did not affect baseline contractile function or the response of control myocytes to isoproterenol, it completely restored the positive inotropic response to isoproterenol in myocytes preincubated in LPS-activated macrophage medium. Release of NO by ventricular myocytes following exposure to activated macrophage medium was detected as an increase in cGMP content in a reporter-cell (RFL-6) bioassay and also as increased nitrite content in myocyte-conditioned medium. Thus, the depressed contractile response of adult rat ventricular myocytes to beta-adrenergic agonists by a 24-h exposure to soluble inflammatory mediators is mediated at least in party by induction of an autocrine NO signaling pathway.

Animals↗
PubMed1993

Coronal-plane transcondylar fracture of the humerus in a child.

A coronal plane fracture of the distal humerus in a child is an uncommon fracture, with the most familiar example being a capitellar fracture. An unusual coronal fracture of the entire distal humeral articular surface (capitellum and trochlea) occurred in a three-year-old boy. This fracture pattern has been described in adults but is believed to be previously unreported in children. This Salter-Harris Type IV fracture was initially misdiagnosed as a lateral humeral condyle fracture. Three years after treatment with open reduction and pin fixation, the distal humerus shows no evidence of growth disturbance.

Bone Wires↗
PubMed1992

Transcriptional regulation in cardiac muscle. Coordinate expression of Id with a neonatal phenotype during development and following a hypertrophic stimulus in adult rat ventricular myocytes in vitro.

The transcriptional regulatory mechanisms in heart muscle that direct cardiac development and allow for a flexible, adaptive response to physiologic stress are not well understood. We demonstrate that a negative regulator of gene transcription termed Id that has been described predominantly in proliferating cell lines and in undifferentiated tissue during growth, is expressed in freshly isolated terminally differentiated adult rat ventricular myocytes, in contrast to most other tissues in the adult rat. Id mRNA expression is regulated in ventricular myocytes during post-natal development, peaking at the transition from hyperplastic to hypertrophic growth at day 17 in the rat, declining subsequently to lower, stable levels in adult myocytes. Although Id mRNA becomes undetectable in adult ventricular myocytes 48 h following isolation in the absence of serum, it can be rapidly reinduced by an alpha-adrenergic agonist, accompanied by increased protein synthesis and the reexpression, in defined media, of the neonatal genes prepro-ANP and skeletal muscle alpha-actin. Thus, the differential regulation of Id during cardiac development, the presence of Id mRNA in normal cardiac myocytes, and its increased expression following a hypertrophic stimulus all suggest a role for this transcriptional regulator in the control of cardiac muscle cell phenotype.

Aging↗
PubMed1992

Recognition and management of digitalis toxicity.

The most important step in the management of toxicity due to any of the cardiac glycosides is its recognition. Despite the development of an accurate clinical assay for serum levels of digoxin greater than 20 years ago, digitalis toxicity remains common and difficult to confirm, even if suspected, due primarily to 2 factors. First, the signs and symptoms of digitalis toxicity, most commonly an abnormal electrocardiogram showing ventricular or atrial arrhythmias, with or without some degree of concurrent atrioventricular block, often also occur in patients with congestive heart failure (CHF) and underlying coronary atherosclerosis who are not receiving a cardiac glycoside. Second, due to digoxin's narrow therapeutic ratio, the marked degree of variability in the sensitivity of individual patients to its toxic effects, and the common problem of obtaining blood samples inappropriately during the early distribution phase following dosing, a serum digoxin concentration often does not serve as a reliable indicator of toxicity. Despite these difficulties in diagnosis, the management of digoxin toxicity has been made much more effective with the widespread availability of F(ab) fragments of anti-digoxin antibodies. This drug provides the clinician with a rapidly acting, safe antidote for all commonly used digitalis preparations. Conventional therapy for digoxin toxicity remains the maintenance of serum potassium levels greater than or equal to 4 mEq/liter, reversal of decompensated CHF or overt myocardial ischemia, attention to serum magnesium levels and the patient's acid-base status, appropriate antiarrhythmics in the event of ventricular arrhythmias, and a temporary pacemaker for high-grade atrioventricular block. Nevertheless, the high specificity and documented safety of the antibody preparation provides a needed safety net for the continuing use of cardiac glycosides as first-line inotropic agents in the modern therapy of chronic CHF.

Digitalis Glycosides↗
PubMed1992

Endothelins. Myocardial actions of a new class of cytokines.

There is growing evidence to support the existence of a dynamic interaction in vivo between cardiac myocytes and adjacent microvascular endothelial cells in the regulation of both cardiac myocyte and possibly endothelial cell phenotype and function. Endothelins may be only one of several endogenous cytokines or autocoids that are released by the cardiac microvascular and/or endocardial endothelium and transported vectorially to adjacent myocytes that could modify cardiac contractile state, perhaps in response to changes in microvascular blood flow. Similarly, cardiac myocytes themselves could release cytokines that could directly affect endothelial cell proliferation or angiogenesis and indirectly elicit or modify the release of endothelium-derived cytokines and autocoids. Thus, in addition to modifying function, endothelial cell-cardiac myocyte interactions may also be of importance in the dynamic events that lead to myocardial wall remodeling and angiogenesis during hypertrophic growth and in the response to cardiac injury.

Amino Acid Sequence↗
PubMed1992

Role of epicardial mesothelial cells in the modification of phenotype and function of adult rat ventricular myocytes in primary coculture.

Adult rat ventricular myocytes undergo a well-documented sequence of phenotypic changes during adaptation to primary culture. However, we observed that coculture of myocytes with a specific subset of nonmyocyte cardiac cells could slow and even reverse the process of adaptation. These nonmyocyte cells were isolated and identified by immunohistochemical and ultrastructural criteria as being of epicardial mesothelial origin. When added to long-term primary cultures of adult ventricular myocytes, epicardial mesothelial cells appeared to induce myofibrillar arrays that were more organized than those seen in noncocultured myocytes; these changes that occurred were concurrent with the appearance of large amplitude contractions and multicellular synchronous beating that was facilitated by gap junctions between myocytes and epicardial mesothelial cells. The changes in morphology and function were accompanied by a marked increase in beta-myosin heavy chain isoform transcription in cocultured myocytes, a return to the ratio of cardiac to skeletal alpha-actin expected in adult rat myocardium, and a much reduced expression of smooth muscle alpha-actin. These changes in myocyte phenotype and function appeared to require epicardial cell-myocyte contact, or close apposition, because media conditioned by epicardial mesothelial cells alone or in coculture had no effect. Thus, these rapid and reversible changes in myocyte ultrastructure, function, and gene expression may provide a useful in vitro model with which to study the mechanism responsible for regulating the plasticity of ventricular myocyte phenotype and the role of specific cell-cell interactions.

Animals↗
PubMed1991

Therapeutic strategies for CHF in the 1990s.

The ideal therapeutic strategy would be to reverse the underlying cause of cardiac dysfunction, such as hypertension or valvular disease, while removing or minimizing those concurrent conditions or factors that tend to exacerbate symptoms. For most patients, vasodilator therapy has been shown to reduce mortality in moderate to advanced CHF.

Adrenergic beta-Antagonists↗
PubMed1991

Leucine-induced amino acid antagonism in rats: muscle valine metabolism and growth impairment.

The deleterious effects of branched-chain amino acid (BCAA) antagonism caused by excess dietary leucine include growth depression and subnormal valine and isoleucine pools. To investigate mechanisms causing these changes, rats were gavage-fed low-protein (9%) diets with or without BCAA supplements, and the metabolism of another BCAA (valine) was measured in incubated rat epitrochlearis muscles. A 10% leucine supplement (HL-10) inhibited growth; growth remained subnormal even when 2.6% isoleucine and 2.4% valine (HLIV-10) were added to the diet. Valine decarboxylation in muscle increased 170-270% in rats fed the HL-10 or HLIV-10 diets, but was still markedly lower than we previously found in muscle of rats fed a 14% protein diet. Valine incorporation into muscle protein as an estimate of protein synthesis was unaffected by any of the BCAA supplements. When a lower (4%) concentration of leucine (without or with 0.16% isoleucine and 0.16% valine) was studied, growth was also suppressed but only if rats had not been preconditioned to 9% protein. Although increased BCAA decarboxylation in muscle caused by excess dietary leucine contributes to low valine and isoleucine pools, abnormal growth appears to be independent of low valine and isoleucine levels and is not reflected in suppression of valine incorporation into muscle protein.

Animals↗
PubMed1991

Endothelin and increased contractility in adult rat ventricular myocytes. Role of intracellular alkalosis induced by activation of the protein kinase C-dependent Na(+)-H+ exchanger.

Endothelin, a 21-amino acid vasoactive peptide, is among the most potent positively inotropic agents yet described in mammalian heart. Having demonstrated that endothelin's inotropic effect is due, in part, to an apparent sensitization of cardiac myofilaments to intracellular calcium, we determined whether this could be due to a rise in intracellular pH (pHi). In isolated adult rat ventricular cells loaded with the H(+)-selective fluorescent probe BCECF, 100 pM endothelin increased contractile amplitude to 190 +/- 26% of baseline and pHi by 0.08 +/- 0.02 (n = 8), whereas 1 nM endothelin increased pHi by 0.13 +/- 0.03 with little further increase in contractility. Amiloride (10(-4)M) prevented the increase in pHi in response to endothelin and reduced the inotropic response by 45%, although the inotropic effect could be readily restored by subsequent NH4Cl-induced alkalinization. Similarly, inhibitors of protein kinase C (H-7 and sphingosine) diminished or abolished the rise in pHi after endothelin superfusion while causing a decline in its inotropic effect comparable with that observed with amiloride. Pretreatment with pertussis toxin, which we have demonstrated results in complete ADP-ribosylation of the alpha-subunits of Go and Gi GTP-binding proteins and abolition of endothelin's positive inotropic effect, only partially reduced the intracellular alkalinization induced by the peptide, suggesting a complex signal transduction mechanism. Thus, the positive inotropic action of endothelin is due in part to stimulation of the sarcolemmal Na(+)-H+ exchanger by a protein kinase C-mediated pathway, resulting in a rise in pHi and sensitization of cardiac myofilaments to intracellular Ca2+.

Alkalosis↗