The sarcoplasmic reticulum does not determine the timecourse of the inotropic response to ß-Adrenoceptor stimulation in rat ventricular cells N.G. Farrar, B. Attilia & J.C. Kentish. Centre for Cardiovascular Biology & Medicine, King's College London, Rayne Institute, St. Thomas's Hospital, London. SE1 7EH

Print abstract Search PubMed for: Farrar NG Attilia B Kentish JC

ß-Adrenoceptor stimulation of cardiac cells leads to the phosphorylation by protein kinase A of a number of proteins, including phospholamban and the calcium release channel (RyR) in the sarcoplasmic reticulum (SR). In isolated channel experiments phosphorylation of the RyR increases the open probability (P_o) of this channel (e.g. Hain et al., 1995), but the functional effect of this in intact cells is not clear. One possibility is that a rise in P_o may accelerate the response to an inotropic intervention such as ß-adrenoceptor stimulation (Eisner et al., 1998). On the other hand, the time-dependence of the loading of the SR with Ca²⁺might be expected to delay the rise of an inotropic response. We investigated whether the SR influences the timecourse of the positive inotropic effect of ß-adrenoceptor stimulation, by selectively inhibiting the SR with thapsigargin (which inhibits the SR Ca²⁺ uptake pump).

Isolated ventricular cells from rats (Wistar, 200 - 250 g, killed by an overdose of pentobarbitone) were superfused with Krebs solution (1 mM Ca²⁺, 23^oC) and stimulated electrically at 0.3 Hz. Cell contractions were recorded by measuring sarcomere length (at 240 Hz) with a video analysis system. We stimulated the b-adrenoceptor pathway by rapidly applying noradrenaline (NA, 10 µM) using a servo-controlled solution switcher. Prazosin (2µM) was present throughout to block œ₁-adrenoceptors. In cells with the SR functional, NA application increased contraction amplitude by 74 ± 23 % (mean ± s.e.m., n=6), and the rise of the inotropic effect could be fitted with an exponential of time constant (t) 35.3 ± 8.2 sec. In cells treated with thapsigargin (3 mM), the contraction amplitude was only 46% of that in untreated cells, but the timecourse of the response to NA application (of 24.8 ± 2.8 s, n=6) was not significantly different (p > 0.05, unpaired t-test) from that in cells with a functional SR. Similar results (of 25.1 ± 4.4 s, n=6) were obtained if the extracellular [Ca²⁺] for the thapsigargin-treated cells was raised to 3 mM, so that their contractions more closely matched (were 87% of) those of untreated cells. Thus the timecourse of the inotropic effect of NA was similar whether or not there was a functional SR.

To investigate whether the timecourse of the inotropic effect of NA is limited by the rate at which intracellular [cAMP] increases, we used flash photolysis of caged cAMP (20 µM) to produce a near-instantaneous increase in intracellular [cAMP]. The photoliberation of cAMP within the cells caused cell contractions to increase by 47 ± 6 % (n=11) with a of 14.9 ± 1.2 s, which was significantly quicker than for cells exposed to NA under the same conditions (P < 0.001, unpaired t-test).

We conclude that, under our conditions, phosphorylation of SR proteins by PKA does not influence the timecourse of the inotropic response to NA. As the response to photolysis of caged cAMP was much faster than that for NA application, a substantial part of the delay in the response to NA is likely to be due to a slow rate at which [cAMP] rises within the cell.

Eisner, D.A., et al. (1998). Cardiovasc Res. 38, 589-604.
Hain, J., et al (1995). J Biol Chem 270, 2074-2081.

We are grateful to the Welcome Trust for financial assistance.