Sian E. Harding

Contamination of a cardiac sarcolemmal preparation with endothelial plasma membrane

Preparation of sarcolemma from whole rabbit heart using the method of Jones et al. (Jones,L.R., Besch, H.R., Fleming, J.W., McConnaughey, M.M. and Watanabe, A.M. (1979) J. Biol. Chem. 254, 530-539) results in a 46-fold purification of the endothelial plasmalemma-specific marker angiotensin converting enzyme. This implies contamination of the sarcolemma with vascular endothelial plasmalemma. During preparation of sarcolemma from sheep heart, using the same method, angiotensin converting enzyme copurified with the general plasma membrane marker (Na+ + K+)-ATPase. The ratio of myocyte to endothelial plasma membrane in the final preparation is therefore similar to that in the whole heart homogenate. Ultrastructural analysis has shown that the myocyte/endothelial surface area is 70:30 in whole cardiac muscle. Comparison of angiotensin converting enzyme activity of an endothelial plasma membrane fraction with that of whole heart sarcolemma suggests an upper limit of 42% for endothelial contamination. Contamination by endothelial plasmalemma was dramatically reduced by preparing sarcolemma from myocytes produced by proteolytic disruption of whole hearts. Following disruption, myocytes were separated from non-muscle cells by sedimentation through 0.5 M sucrose. Sarcolemma prepared from sheep cardiac myocytes had approximately 15-fold less angiotensin converting enzyme activity than whole sheep heart sarcolemma but comparable ouabain-inhibitable (Na+ + K+)-ATPase activity.

An antiadrenergic effect of adenosine on guinea-pig but not rabbit ventricles

The antiadrenergic effect of adenosine was investigated using isolated guinea-pig heart and guinea-pig and rabbit papillary muscle. Adenosine, 15 microM, completely abolished the increased tension stimulated by 0.1-1.0 nM isoprenaline in Langendorff-perfused guinea-pig hearts. With guinea-pig papillary muscles, adenosine decreased by 40% the increased force stimulated by 1-10 nM isoprenaline. When 5 microM 2-chloroadenosine was used in conjunction with 1 unit ml-1 adenosine deaminase, a complete inhibition of the isoprenaline-stimulated tension was seen in guinea-pig papillary muscles. The antiadrenergic effect of 2-chloroadenosine was blocked by 8-phenyltheophylline. In rabbit, there was little effect of 2-chloroadenosine (plus deaminase) on isoprenaline-stimulated tension. (-)-N6 (R-phenylisopropyl)-adenosine (PIA) had no effect on basal or isoprenaline-stimulated adenylate cyclase activity of guinea-pig or rabbit sarcolemmal membranes. We conclude that the antiadrenergic effect of adenosine is mediated by A type receptors and is seen in guinea-pig but not rabbit. Production of adenosine by superfused papillary muscle may obscure the effect of added adenosine. We find no evidence that the antiadrenergic effect is mediated by inhibition of adenylate cyclase.

Pertussis toxin reduces the antiadrenergic effect of 2-chloroadenosine on papillary muscle and the direct negative inotropic effect of 2-chloroadenosine on atrium

2-Chloroadenosine reduced the contractile tension of guinea-pig atria directly, and inhibited the increase in tension produced by beta-adrenergic stimulation of guinea-pig papillary muscle. Both effects were reduced by 8-phenyltheophylline, a competitive antagonist at extracellular P1-purinoceptors. Treatment of guinea-pigs with pertussis toxin reduced the sensitivity of both atria and ventricles to 2-chloroadenosine. Atria were significantly affected after treatment with 125 micrograms/kg toxin, but not 100 micrograms/kg. 60 micrograms/kg toxin had no effect on the sensitivity of the ventricles, but 100 and 125 micrograms/kg significantly decreased the antiadrenergic effect of 2-chloroadenosine. We conclude that both the direct and antiadrenergic effects are mediated by an inhibitory guanine nucleotide binding protein.

GTP-independent stimulation of rabbit heart adenylate cyclase by isoproterenol at physiological ATP concentrations.

SummaryIsoproterenol increased the activity of the adenylate cyclase of rabbit heart sarcolemmal membranes in the absence of added GTP. ATP, the ATP-regenerating system, and the sarcolemmal membrane preparation were eliminated as possible sources of contaminating GTP. Isoproterenol-stimulation increased as ATP was raised. At 0.5 mM ATP, isoproterenol increased activity by 19% whereas at 5 mM ATP isoproterenol increased activity by 121%. There was no change in basal activity between 0.5 and 5 mM ATP. Stimulation by Gpp(NH)p and NaF increased slightly between 0.5 and 5 mM ATP; stimulation by KCl was unaffected. GTP does not activate cyclase d. GTP does not activate cyclase to the same extent as Gpp(NH)p even though the two act at the same site on Ns (the stimulatory guanine nucleotide-binding protein). GTP decreased cyclase activation by Gpp(NH)p in a concentration-dependent fashion when the two were added to the assay simultaneously. Increasing ATP from 0.5 to 5 mM did not reduce activation by Gpp(NH)p when both were added simultaneously to the assay. This suggests that ATP does not interact with the same site as Gpp(NH)p. ATPγS, an analogue of ATP which irreversibly thiophosphorylates proteins, did not irreversibly support activation by isoproterenol. The effect of ATP in supporting isoproterenol stimulation is not, therefore, thought to be due to phosphorylation of a protein.

Inhibition of rabbit cardiac adenylate cyclase by theophylline

Theophylline inhibits basal adenylate cyclase activity as well as cyclase stimulated by sodium chloride, sodium fluoride, GTP or 5′‐guanylimidodiphosphate. This inhibition is dose‐dependent and shows non‐competitive inhibition, with respect to MgATP. The presence of adenosine deaminase does not alter the effect of theophylline. The inhibition produced by theophylline is not additive with that due to 2′‐deoxyadenosine 3′‐monophosphate (a P‐site agonist). It is suggested that theophylline may act at the P‐site to reduce adenylate cyclase activity.

Myocardial Cell Abnormalities in Heart Failure: Experience from Studies on Single Myocytes

In the majority of patients with heart failure the underlying cause is one of myocardial dysfunction, which may in itself be either primary in origin or secondary to diseases of the coronary arteries or of the heart valves. In terms of cellular structure and function, myocardial failure can be due to abnormalities in the myocytes themselves or to abnormalities in the surrounding extracellular matrix. Histological studies in ischemic and dilated cardiomyopathy have revealed a spectrum of histological changes, including collagen accumulation, fibrosis, loss of myofilaments, myocyte slippage, myocyte hypertrophy, and cell death [1–3]. Although these changes alone have been postulated to produce progressive myocardial failure due to disruption of ventricular geometry [4], there is also evidence of a substantial functional impairment of the cardiac myocytes. In this chapter we review the evidence for the role played by this impairment of myocyte function, which has been identified from studies using isolated cardiac myocytes.