Effects of phorbol ester on contraction, intracellular pH and intracellular Ca2+ in isolated mammalian ventricular myocytes.

Abstract

SUMMARY1. We have investigated the actions of certain phorbol esters on the intracellular pH, intracellular Ca2+ and contractility of isolated rat and guinea-pig cardiac myocytes. Intracellular pH was measured using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and intracellular Ca2+ was measured using Fura-2.2. Application of the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (also called phorbol 12-myristate 13-acetate) (TPA) (which activates protein kinase C) to rat cardiac myocytes significantly increased cell shortening by 116 + 34% (n = 8) (p < 002). The rate of change of cell length during contraction (i.e. + dL/dt) increased from 67-2 + 817 gim/s to 127-7 + 14-1 gm/s (n = 7). The rate of change of cell length during relaxation (-dL/dt) increased from 55-8 + 7X4 gm/s to 1 18-9 + 1241 gcm/s (n = 7). Time to peak shortening was unchanged.3. Application of 4a-phorbol 12,13-didecanoate, which does not activate protein kinase C, did not affect rat myocyte contractility. An insignificant decrease in contractility (by 7-5 + 7'5%) was observed (n = 5). The positive inotropic effect of TPA may therefore be evoked through an activation of protein kinase C.4. In rat myocytes we have measured the changes of pHi and contractility (cell shortening) during an alkalosis and acidosis induced by exposure to and subsequent removal of NH4Cl both in the presence and absence of TPA. Recovery times from an acid load were significantly (p < 005) enhanced by 1541+ 69 % (n = 13) in the presence of TPA. Recovery times of cell shortening were also more rapid (p < 005) by an average of 59-1 + 10-6 % (n = 5) in the presence of TPA. Recovery times were unchanged in the presence of 4-phorbol 12,13-didecanoate (wirhich does not activate protein kinase C).5. Since pHi recovery of an isolated myocyte from an acid load is partially inhibited by the presence of 1 mM-amiloride and inhibited by removing extracellular Na+ then it is suggested that, like pHi regulation in sheep heart Purkinje fibres, pH1 recovery in rat cardiac ventricular myocytes is mainly through sarcolemmal Na+-H+ exchange. We suggest that in the presence of TPA the Na+-H+ exchange is stimulated. (diastolic) [Ca21]. Fura-2 ratios increased by 21+4%(n = 5). We conclude that some of the positive inotropic effect produced by phorbol esters which activate protein kinase C is caused by an increase in systolic Ca2+.

Endothelin and angiotensin II stimulation of Na+-H+ exchange is impaired in cardiac hypertrophy.

“…It is well established that the effects of the vasoactive peptides ET-1 and angiotensin II may vary with the preparation which is studied and with the stage of development. In intact isolated heart preparations, both positive and negative inotropic effects of ET-1 and angiotensin II have been reported in adult rabbits and rat hearts (10,11,16,29,33). These conflicting responses may be related to the effects of these vasoactive peptides on coronary vasomotor tone, thus confounding the assessment of direct myocardial actions.…”

“…In the present experiments, ET-1 and angiotensin II failed to cause intracellular alkalinization in the presence of a selective protein kinase C inhibitor in normal cells. MacLeod and Harding (29) have reported previously that the recovery from an acute acid load in isolated rat myocytes is partially inhibited by amiloride or by removing extracellular Na ϩ . They observed that exposure to phorbol ester 12-myristate 13-acetate (which activates protein kinase C) exaggerated the changes in intracellular pH and associated changes in cell shortening in normal rat and guinea pig myocytes in response to NH 4 Cl challenge, which suggests the stimulation of Na ϩ -H ϩ exchange.…”

“…Upon washout of extracellular NH 4 Cl, intracellular acidosis is created as internal NH 3 leaves the cell, causing the intracellular retention of H ϩ . Previous studies have clarified that the initial rate of recovery from this intracellular acidosis depends predominantly on forward Na ϩ -H ϩ exchange (12,29). In additional experiments, the same protocol was done in SNARF-1 loaded hypertrophied (n ϭ 4) and control (n ϭ 8) myocytes in the presence and absence of 10 nM angiotensin II.…”

Molecular Basis for Angiotensin II-Induced Increase of Chloride/Bicarbonate Exchange in the Myocardium

“…PMA stimulates sarcolemmal NHE of isolated ventricular myocytes. 36 To rule out an effect of NHE in the observed PMA-induced increase of AEfl activity, anion exchange assays were repeated under conditions where NHE was fully blocked (amiloride, 1 mmol/L). In the presence of amiloride, steady-state pH i decreased slightly after PMA addition (7.29Ϯ0.03 and 7.23Ϯ0.01 before and after PMA, respectively, nϭ3), but NHE blockade did not affect the PMAinduced increase of AE3fl activity, 2.6Ϯ0.1 and 3.8Ϯ0.2 mmol/L · min Ϫ1 , before and after PMA, respectively (48Ϯ3% of increase, nϭ3, PϽ0.05).…”

Raloxifene acutely suppresses ventricular myocyte contractility through inhibition of the L‐type calcium current

“…Experiments were performed on single left ventricular myocytes isolated from adult male guinea pigs (550-750 g) by enzymatic digestion as previously described (MacLeod & Harding, 1991). We chose to study male, rather than female, animals to avoid the potential problems associated with varying expression of the ER that occurs throughout the oestrus cycle.…”