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In Chinese hamster ovary cells stably expressing the cloned human cholecystokinin (CCK)B/ gastrin receptor, cholecystokinin octapeptide (CCK-8) evoked increases in [Ca2+]i monitored by digitized video imaging of fura-2 fluorescence ratios. At concentrations around 10 pM, CCK-8 elicited [Ca2+]i oscillations, which were blocked by elimination of extracellular Ca2+, by a phospholipase C inhibitor, U-73122, by a protein kinase C inhibitor, H7, as well as by phospholipase A2 (PLA2) inhibitors, ONO-RS-082 and aristolochic acid. At higher concentrations, CCK-8 induced a single biphasic [Ca2+]i rise consisting of a large peak followed by a lower sustained plateau, while the response turned into [Ca2+]i oscillation when the extracellular Ca2+ was eliminated or a PLA2 inhibitor was included. CCK-8 stimulated the release of arachidonic acid, and this was inhibited by aristolochic acid. Arachidonic acid caused an increase in [Ca2+]i which was dependent upon extracellular Ca2+. These results suggest that the activation of PLA2 might be involved, at least in part, in the Ca2+ influx that maintains the sustained plateau phase of [Ca2+]i as well as the [Ca2+]i oscillation when CCKB receptors are stimulated.

We determined the effect of 1-cis diltiazem, the enantiomer of diltiazem (d-cis isoform), on the energy metabolism of isolated guinea pig hearts during ischemia-reperfusion. We used 31P-NMR to measure the high-energy phosphate content and intracellular pH (pHi) during global ischemia for 30 min followed by reperfusion for 30 min. Before ischemia, the left ventricular developed pressure (LVDP) was reduced less by 10 microM l-cis diltiazem than by 3 microM diltiazem or 500 nM nifedipine. However, 10 microM l-cis diltiazem preserved the intracellular ATP content during ischemia and reperfusion, reduced the end-diastolic pressure increase during ischemia and reperfusion, and restored LVDP after reperfusion. Nifedipine at 50 nM, which reduced the LVDP more than 10 microM l-cis diltiazem, showed no cardioprotective effect. Ten micromolar l-cis diltiazem and 3 microM diltiazem, but neither 50 nor 500 nM nifedipine, reduced the pHi decrease that occurred 25 or 30 min after the onset of ischemia. Therefore, l-cis diltiazem has a cardioprotective effect on ischemic and reperfused myocardium and is less cardiodepressive than diltiazem and nifedipine. The effect of l-cis diltiazem during ischemia and reperfusion involves energy preservation, which is probably independent of its Ca2+-channel blocking action.

l-cis Diltiazem, an optical isomer of diltiazem, protects against myocardial dysfunction in vitro, whereas its Ca2+ channel blocking activity is about 100 times less potent than that of diltiazem. However, there is no evidence that l-cis diltiazem actually protects against ischemia/reperfusion injury in vivo. To assess this, we employed an anesthetized rabbit model, where the left circumflex artery was occluded for 15 min and reperfused for 360 min. Treatment with diltiazem before and during ischemia (bolus 200 microg/kg and 15 microg/kg per minute for 25 min, i.v.; 575 microg/kg total) showed slightly depressed hemodynamic parameters, while l-cis diltiazem (1150 microg/kg) had no effect. Treatment with l-cis diltiazem produced a high recovery of the thickening fraction and limited the infarct size in a dose-dependent manner. Furthermore, the treatment with l-cis diltiazem (1150 microg/kg) or diltiazem (575 microg/kg) 5 min before reperfusion also limited the infarct size, but not after reperfusion. These results suggest that l-cis diltiazem affects some events in the onset of reperfusion, independently of Ca2+-channel-blocking action. Our observations are the first to show that l-cis diltiazem demonstrated its cardioprotective action in the ischemic rabbit heart in vivo.

To examine the contribution of beta-adrenoceptor (beta AR) downregulation to desensitization of beta ARs by chronic administration of a beta AR agonist, we compared the adenylyl cyclase (AC) activities in two kinds of cardiac ventricular membranes with decreased available beta ARs: one was derived from rats infused with a selective beta 1 AR agonist, T-0509 [(-)-(R)-1-(3,4-dihydroxyphenyl)- 2-[(3,4-dimethoxyphenethyl)-amino]ethanol hydrochloride], in vivo (40 micrograms/kg/hr, s.c. for 6 days); and the other was obtained from treatment of control membranes with an irreversible beta AR antagonist, bromoacetyl alprenolol methane (BAAM). T-0509 infusion decreased the densities of beta 1 ARs and beta 2 ARs by 26% and 32%, respectively, and reduced the maximal isoproterenol-stimulated AC activity by 53%. The amount of Gs alpha and Gi alpha proteins in the membranes was not significantly changed by T-0509 infusion. To make preparations that mimic the T-0509-induced downregulation, we treated the control membranes with 100 nM BAAM in vitro. The BAAM treatment decreased the Bmax value of [125I]iodocyanopindolol for beta 1 ARs and beta 2 ARs by 29% and 36%, respectively, whereas it reduced the maximal effect of isoproterenol on AC activity only by 37%. These results suggest that downregulation of beta ARs cannot fully account for the desensitization by chronic treatment of T-0509 and that other mechanism(s) can play a significant role in the loss of responsiveness.

The effects of diltiazem on the cerebral blood flow and cerebrovascular spasm were studied in pentobarbital anesthetized animals. In Rhesus monkeys, the common carotid and internal carotid blood flow were measured by an electromagnetic flowmeter. Diltiazem (10-300 micrograms/kg, i.v.) dose-dependently increased both the common carotid and internal carotid blood flow, and the increase in internal carotid blood flow persisted for a longer period than that in the common carotid blood flow. In dogs, regional blood flow in the cerebral cortex (rCBF) was measured by means of the hydrogen gas clearance method. Diltiazem (20 micrograms/kg/min, i.v.) increased rCBF by about 20% of the control during the infusion, and the increase in rCBF was still continued 40 min after the infusion was stopped. In cats, the basilar artery was exposed by craniotomy through the transcervico-transclival approach, and vasospasm was induced by topical administration of 5-HT, PGF2 alpha and incubated blood. Diltiazem, either applied topically to the artery (100 micrograms/ml) or infused continuously into the femoral vein (20 or 40 micrograms/kg/min), suppressed the vasoconstriction evoked by the spasmogen.