A K(ATP) channel opener inhibited myocardial reperfusion action potential shortening and arrhythmias.

Low concentrations of certain K(ATP) channel openers have been reported to exert a moderate inhibitory effect on arrhythmias during post-ischaemic early myocardial reperfusion, but the accompanying effects on the time course of changes in action potentials in intact hearts have not yet been studied. We report that in rat isolated hearts, reperfusion following 10 min of regional no-flow ischaemia was associated with both an acute, marked, but transient, shortening of ventricular repolarisation (by 63%) during reperfusion, and a high incidence (90%) of ventricular tachyarrhythmias. The K(ATP) channel opener Ro 31-6930 [2-(6-cyano-2,2-dimethyl-2H-1-benzopyran-4-yl)-pyridine 1-oxide], delivered prior to ischaemia at a relatively low concentration (0.5 microM), significantly reduced the incidence and duration of reperfusion arrhythmias, and prevented the associated acute action potential shortening during reperfusion, each in a glibenclamide (1 microM)-sensitive manner (P<0.05, n=10-15 hearts). This was associated with a moderate and non-arrhythmogenic action potential shortening during ischaemia (a potentially "cardioprotective" effect). However, these data highlight the potential harm these drugs may cause, since a higher concentration of Ro 31-6930 caused marked shortening of action potentials and significant pro-arrhythmia during ischaemia.

Do KATP channels open as a prominent and early feature during ischaemia in the Langendorff-perfused rat heart?

The objective was to investigate whether myocardial adenosine triphosphate-sensitive K+ (KATP) channels open during the first 10 min of regional ischaemia in Langendorff-perfused rat hearts. Changes in monophasic action potentials and arrhythmias were studied during myocardial ischaemia in both the presence and absence of pharmacological KATP modulation. Ligation of the left main coronary artery for 10 min did not shorten the action potential duration (APD). The APD50 and APD80 (15.5 +/- 1.0 and 38.1 +/- 2.3 ms, respectively [mean +/- S.E., n = 15 hearts], immediately prior to ligation) increased transiently during the first 4 min of ligation (by 160 and 79% respectively, P < 0.05), before returning to pre-ligation values, but without a significant below-baseline-shortening. The cardiac electrogram showed no accompanying ventricular tachyarrhythmia (VT). These results raised the possibility that the myocardial KATP channels had not opened during the ligation. The KATP opener Ro 31-6930 (0.5 and 5 microM) shortened the APD50 and APD80 during coronary ligation, to significantly below both their control and pre-occlusion values (P < 0.05), and caused a concentration-dependent increase in both the incidence and duration of VT during the ligation. Ro 31-6930 at 5 microM also shortened APD50 and APD80 even before ligation (by 50 and 62% respectively, P < 0.05), and abolished the normal APD-lengthening seen during ischaemia. The KATP blocker glibenclamide (1 microM) abolished both the APD-shortening and pro-arrhythmic effects of the KATP opener, both before and during coronary ligation, yet when delivered on its own, at the same concentration which abolished the effects of KATP activation, it had no significant effect on the APD changes seen during the coronary ligation alone. These results suggest that, in Langendorff-perfused rat hearts in the absence of drugs, KATP channels do not open during early myocardial ischaemia.

Effect of sodium nitroprusside and L-arginine methyl ester on rat hearts stored at 4 degrees C for 24 h.

1. This study examined the effects of altering nitric oxide levels with sodium nitroprusside or L-arginine in rat hearts stored hypothermically.2. Hearts were microperfused at 4 degrees C for 24 h with a modified Krebs-Henseleit buffer (KHB) that contained either sodium nitroprusside, L-arginine, L-arginine methyl ester or dexamethasone.3. After hypothermic storage, hearts were rewarmed to 37 degrees C with KHB alone or KHB containing sodium nitroprusside or L-arginine. Cardiac function was then assessed in either Langendorff mode or working heart mode.4. Compared with values from fresh unstored hearts, hypothermic stored hearts showed a significant decrease in coronary flow and left ventricular developed pressure when the stored hearts were perfused in Langendorff mode. These hearts also produced less aortic flow and cardiac output when perfused in the working mode.5. Hearts hypothermically microperfused with buffer containing either L-arginine or sodium nitroprusside and then reperfused in the Langendorff mode with untreated KHB buffer had the highest left ventricular developed pressure and coronary flow values. Aortic flow and cardiac output were also higher in these hearts.6. In all groups of stored hearts, the concentrations of both ATP and creatine phosphate were significantly low, when compared with values from freshly isolated hearts. Addition of dexamethasone to the buffer either during storage or during reperfusion had no beneficial effect on high-energy phosphate loss or cardiac performance of stored hearts.7. This study showed that the addition of nitric oxide donors to storage buffer significantly improves cardiac function on normothermic reperfusion. The improved functional recovery is unrelated to the high-energy phosphate content of these hearts.

Mast cell amines and inosineinduced vasoconstriction in the rat hind limb.

Under certain circumstances injected inosine causes a net vasoconstrictive effect on the arterioles, which has been attributed to 5-hydroxytryptamine (5HT) released in response to adenosine type 3 (A(3)) receptor stimulation of mast cells residing in the adventitia. We have sought further evidence for this hypothesis using blood vessels of the rat hind limb perfused in vitro at constant rate with a gelatin-containing physiological salt solution. Injection of inosine (2.7 mg) caused a rise in perfusion pressure, which was only slightly increased by inclusion of N-nitro-L-arginine methyl ester (100 muM) in the perfusate. Inclusion in the perfusate of cyproheptadine (1 muM), compound 48 80 (1 mug ml), 8-phenyltheophylline (1 muM) or 8-cyclopentyl-1,3 dipropylxanthine (0.1 muM) greatly reduced the pressor response to inosine. The pressor effect of injected 5HT (400 mug) was abolished by pre-treatment with cyproheptadine, but not by pre-treatment with compound 48 80. These results suggest that the net pressor response to injected inosine was mainly the result of an A(1) receptor-mediated release of 5HT, most probably from mast cells. No evidence was found for an involvement of A(3) receptor stimulation.

Some cyclic nucleotides reduce phenylephrine-induced and phorbol ester-induced constriction of the rat anterior mesenteric artery.

1. Vasoconstrictor responses to phenylephrine (PE) and to 12-deoxyphorbol 13-phenyl-acetate in the rat isolated perfused anterior mesenteric artery were inhibited by pretreating the artery with isoprenaline (ISOP), sodium nitroprusside or 8-bromoguanosine 3':5'-cyclic monophosphate, but not with 8-bromoadenosine 3':5'-cyclic monophosphate. 2. The inhibitory effect of ISOP toward the response to PE was less prominent after pretreating the artery with a selective inhibitor of cyclic GMP-dependent kinase. 3. The vasorelaxant effect of ISOP was enhanced by pretreating the artery with an inhibitor of nitric oxide synthase.

Effect of protein kinase C inhibitors and 2,3-butanedione monoxime on the long-term hypothermic preservation of isolated rat hearts.

1. This study examines the protective effect of staurosporine, chelerythrine, Ro 31-8220 and 2,3-butanedione monoxime in rat hearts during hypothermic storage. 2. Hearts were microperfused at 4 degrees C for 24 or 48 h with a storage buffer that in some cases contained one of these protein kinase C inhibitors either alone or in combination with 2,3-butanedione monoxime. After hypothermic storage, hearts were rewarmed to 37 degrees C with Krebs-Henseleit buffer. Cardiac function was then assessed in either Langendorff mode or working heart mode. 3. Compared with values from fresh non-stored hearts, hypothermic stored hearts showed a significant decrease in both coronary flow and left ventricular developed pressure when the stored hearts were reperfused in Langendorff mode. The decrease in coronary flow and left ventricular developed pressure was more pronounced in hearts stored for 48 h than in those stored for 24 h. 4. Hearts stored for 24 or 48 h, with or without the protein kinase C inhibitors, and then perfused in working mode generated less aortic flow and less cardiac output than fresh unstored hearts. 5. Hearts preserved in solutions containing staurosporine, chelerythrine, Ro 31-8220 or 2,3-butanedione monoxime had significantly higher left ventricular developed pressure values on reperfusion than hearts stored without any such drug. 6. Addition of 2,3-butanedione monoxime to a storage buffer containing either staurosporine, chelerythrine or Ro 31-8220 further improved left ventricular developed pressure, aortic flow and cardiac output values in these stored hearts. The group of hearts stored in a buffer containing 2,3-butanedione monoxime and chelerythrine gave the highest left ventricular developed pressure value seen during reperfusion. 7. The ATP and creatine phosphate concentrations of hearts stored in buffer alone were significantly lower than those of fresh unstored hearts, irrespective of the duration of storage. ATP concentrations were better preserved in hearts stored in a buffer containing 2,3-butanedione monoxime or/and one of the protein kinase C antagonists than those stored without such antagonists. A positive correlation was found between peak cardiac output values and the concentrations of combined high-energy phosphates in various groups of stored and reperfused hearts. 8. The present study showed that inhibition of protein kinase C during long-term hypothermic storage significantly increased high-energy phosphate concentrations and also improved contractile function during reperfusion.

Rat intestinal mast cell amines are released during nitric oxide synthase inhibition in vitro.

Inhibition of nitric oxide synthase increases microvascular permeability in rat small intestinal villi. To determine the mechanism(s) whereby this occurs we have perfused the vasculature of rat isolated small intestines with a gelatin-containing physiological salt solution. Inclusion of N-nitro-L-argintne methyl ester (L-NAME, 100 muM) or indomethacin (1 muM) in the perfusate increased leakage of injected colloidal carbon into microvessel walls. Pre-treatment with sodium nitroprusside (10 muM) significantly reduced the effects of both L-NAME and indomethacin, whereas carbacyclin (1 muM) only reduced the effects of indomethacin. PD151242 (1 muM) showed some antagonism towards the effects of L-NAME, but nordihydroguaiaretic acid (3 muM) was inactive. Pre-tment with cyproheptadine (10 muM) reduced the effects of both L-NAME and indomethacin, and also significantly reduced background (control) colloidal carbon leakage. Small intestines from polymixin B-treated rats showed significantly reduced colloidal carbon leakage in response to L-NAME. This suggests that the leakage-enhancing effects of both L-NAME and indomethacin in this preparation may be mediated by mast cell-derived amines.

Degranulation of rat omental mast cells by A(1) receptor agonists in vitro.

The haemodynamic effects of adenosine are thought to result in part from a release of mast cell amines via A3 receptor stimulation. To investigate the nature of the receptors involved in adenosine-induced mast cell degranulation in the rat isolated omentum we have used adenosine analogues with varying specificities as activators of the A(1), A(2) and A(3) receptors, and antagonists with differing specificities for A(1) and A(2) receptors. Analogues which act predominantly as A(1) (e.g. N(6)-cyclopentyladenosine) or as mixed A(1)/A(2) receptor agonists (e.g. adenosine, inosine, 5'-(Nethylcarboxamido) adenosine) caused mast cell degranulation, whereas a predominantly A3 receptor agonist (IB-MECA) was inactive. Pre-treatment of the omentum with the A(1)/A(2) receptor antagonist 8-phenyltheophylline or with the more specific A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine significantly reduced agonist-induced degranulation. Pre-treatment with disodium cromoglycate or with BN52021 also reduced degranulation of mast cells in response to N(6)-cyclopentyladenosine. In the rat isolated omental mast cell we conclude that degranulation is an indirect result of A(1) receptor stimulation. Platelet-activating factor release appears to mediate at least part of the degranulation.

Inhibition of NO-synthase and degranulation of rat omental mast cells in vitro.

Mast cell amines, platelet-activating factor (PAF), thromboxanes and leukotrienes have been shown to be released during nitric oxide-synthase inhibition in the rat intestine. Mast cells in rat isolated omentum (OMCs) or isolated from the rat peritoneal cavity (PMCs) have been used here to investigate the relationship(s) between these agents. N-nitro-L-arginine methyl ester (L-NAME, 100 muM) caused some degranulation of OMCs, but no enhancement of histamine release from PMCs. PAF (5 muM) and U46619 (1 muM) degranulated OMCs and enhanced histamine release from PMCs. Pre-treatment of the omentum with BN52021 (10 muM) inhibited degranulation of OMCs in response to L-NAME, PAF or U46619. Pretreatment with 1-benzylimidazole (5 or 50 muM) inhibited the effect of L-NAME but not that of PAF. Indomethacin (1 muM) or sodium nitroprusside (10 muM) also inhibited the effects of L-NAME, but nordihydroguaiaretic acid (30 muM) did not. In PMCs BN52021 inhibited PAF-induced, but not U46619-induced, release of histamine. These results suggest that inhibition of nitric oxidesynthase in the omentum by L-NAME allows thromboxanes to release PAF, which in turn degranulates and releases histamine from OMCs.

Long-term preservation of the rat isolated heart with staurosporine and 2,3-butanedione monoxime.

The present study was designed to investigate the effectiveness of staurosporine and 2,3-butanedione monoxime (BDM) in preserving cardiac function of long-term hypothermic-stored hearts. Rat isolated hearts were perfused very slowly at 4 degrees C for 16 hr with a storage buffer solution containing staurosporine and BDM. Heart functions were then examined during 2 hr of normothermic reperfusion. Isovolumetric left ventricular-developed pressure (LVDP), its differential, heart rate, and coronary flow were measured in 5 groups of hearts: controls (fresh unstored hearts), stored drug-free hearts, stored staurosporine-treated hearts, stored BDM-treated hearts, and stored BDM + staurosporine-treated hearts. Hearts that had been perfused with staurosporine or BDM during hypothermic storage attained LVDP values that were 37% or 70%, respectively, of that shown by the control group. Hearts perfused without any drug in the storage buffer attained an LVDP value that was 20% of the control value. Heart rates of stored and then normothermically reperfused hearts were lower than, but not significantly different from, values in the control group. Coronary flow values in all stored hearts were significantly lower than the control values. Thus, BDM, and to a lesser extent staurosporine, applied during prolonged hypothermic storage improved cardiac function during normothermic reperfusion.

Possible bi-directional link between ET(A) receptors and protein kinase C in rat blood vessels.

Possible links have been investigated between activation of protein kinase C (PKC) and endothelin (ET) production by small blood vessels. Perfusion pressures were recorded from rat isolated mesenteric artery, with or without the small intestine attached, before and after addition to the perfusate of either ET-1, ET-3 or the PKC activator 12-deoxyphorbol 13-phenylacetate (DOPPA). Rises in perfusion pressure in response to ET-1 (10(-8) M)or DOPPA (10(-6) M) were reduced significantly by pre-treatment with either the ET(A) receptor antagonist PD151242 (10(-6) M) or the PKC inhibitor Ro 31-8220 (10(-6) M). ET-3 (10(-8) M) had a significant, albeit small, effect only when the gut was still attached to the mesentery. Inthis latter preparation ET-1 and DOPPA increased the permeability of villi microvessels to colloidal carbon in the perfusate. This effect of DOPPA was reduced by pre-treatment with either PD151242 or Ro 31-8220, but the effects of ET-1 were reduced significantly only by Ro 31-8220. ET-3 (10(-8) M) was without effect. The results suggest a possible bi-directional link between ET(A) receptors and PKC in the intestinal vasculature.

Rate of perfusion modulates colloidal carbon leakage from rat intestinal microvessels in vitro.

In order to investigate the effects of varying the rate of flow on endothelial integrity the rat isolated small intestinal vasculature was perfused at 1, 5, 10 or 20 ml/min with a gelatin-containing physiological salt solution (GPSS), followed by an injection of colloidal carbon suspension (CC). Significantly greater microvascular CC leakage occurred at 1 or 5 ml/min than at 10 or 20 ml/ mitt. CC leakage at the two slower rates of flow was reduced by adding red blood cells to the GPSS, suggesting that the microvascular endothelium became hypoxic when perfused with GPSS at 1 or 5 ml/min. After perfusion at 20 ml/min with GPSS containing resiniferatoxin (1 muM) or 5-hydroxytryptamine (100 muM), CC leakage was significantly lower than after similar perfusion at 10 ml/min. Two nitric oxide (NO) synthesis blockers, N-nitro-L-arginine methyl ester (L-NAME, 100 muM) and methylene blue (20 muM), and an NO scavenger CPTIO (100 muM) each increased CC leakage. This suggests that NO was being produced at perfusion rates of 10 or 20 ml/min. Sodium nitroprusside (10 muM), 8-bromo-cGMP (100 muM) and BN52021 (10 muM) each significantly reduced CC leakage in the presence of L-NAME.

Vasoconstriction in rat isolated mesentery and small intestine in response to various activators of protein kinase C.

The vasculature of rat isolated mesentery and small intestine was perfused with a gelatin-containing physiological salt solution (GPSS). When 5-hydroxytryptamine (5HT, 1 x 10(-4) M), or the calcium ionophore A23187 (1 x 10(-4) M), or 12-deoxyphorbol 13-phenylacetate (DOPPA, 1 x 10(-6) M), or 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPAA, 1 x 10(-6) M) or thymeleatoxin (TMX, 1 x 10(-6) M) was added to the GPSS for 5 min there was a gradual rise in perfusion pressure, whereas resiniferatoxin (RFX, 1 x 10(-6) M) was without effect. Pre-treatment of the tissue with the protein kinase C (PKC) inhibitor Ro 31-8220 (1 x 10(-6) M) significantly reduced the rise in perfusion pressure in response to 5HT, DOPPA, DOPPAA and TMX, but not that to A23187. Platelet-activating factor (PAF, 5 x 10(-6) M) caused an almost immediate but transient rise in perfusion pressure, followed by a more gradual rise, neither response being blocked by Ro 31-8220. When blood vessels of the mesentery alone were perfused with gelatin-free PSS, PAF caused a transient rise in perfusion pressure, but with no subsequent gradual rise over 5 min. After Ca(2+)-depletion this transient response was also absent. In contrast, when blood vessels were perfused with gelatin-free PSS, DOPPA and TMX still caused gradual rises in perfusion pressure, which were totally abolished by Ro 31-8220. TMX had no effect at all when the tissue was depleted of Ca2+, whereas the response to DOPPA was only partially reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of protein kinase C activity may increase microvascular permeability to colloidal carbon via alpha-isoenzyme.

The vasculature of the isolated mesentery and small intestine was perfused with a gelatin-containing physiological salt solution in vitro. Various phorbol-related compounds that are known to have different affinities for the protein kinase C (PKC) isoenzymes, and bradykinin (BK), were tested for their ability to cause the microvascular endothelium to become permeable to injected colloidal carbon (CC). Phorbol 12,13-dibutyrate (PDB), 12-deoxyphorbol 13-phenylacetate (DOPPA), thymeleatoxin (TMX), and resiniferatoxin (RFX), each at a concentration of 1 microM, were found to increase permeability. Pretreatment with the PKC inhibitor Ro 31-8220 (1 microM) significantly reduced the response to all of these compounds. Indomethacin (1 microM), on the other hand, reduced only the effect of RFX. 12-Deoxyphorbol 13-phenylacetate 20-acetate (DOPPAA) (1 microM) and BK (10 microM) did not increase CC leakage. These results suggest that the Ca(2+)-dependent PKC alpha-isoenzyme was involved in the increase in endothelial permeability. BK does not appear to stimulate PKC activity in this experimental situation.

Effect of pre-treating rat atria with potassium channel blocking drugs on the electrical and mechanical responses to phenylephrine.

Action potential duration (APD) and systolic developed tension (SDT) were recorded from electrically paced rat atria before, during and after treatment with phenylephrine in vitro. Phenylephrine caused a prolongation of APD and an increase in SDT, the magnitude of which depended upon the frequency of pacing of the atria. Effects very similar to those elicited by phenylephrine were produced by exposing the atria to 4-aminopyridine, 3,4-diaminopyridine, CsCl, amantadine or sparteine. Atria that had been pre-treated with any of the latter five compounds showed a diminished responsiveness when subsequently treated with maximally effective concentrations of phenylephrine, and vice versa. These findings are consistent with blockade of potassium ion channels being responsible for the observed responses to all of the above agents.

Lectin-induced increase in microvascular permeability to colloidal carbon in vitro may involve protein kinase C activation.

Two plant lectins, wheat germ agglutinin (WGA) and concanavalin A (Con A), which are known to bind to endothelial cells (ECs), were found to increase the leakage of colloidal carbon (CC) into the walls of microvessels in the villi of rat small intestine, when added to a gelatin-containing perfusate (GPSS) at a concentration of 10 micrograms/ml. Pretreatment of the microvessels with the protein kinase C (PKC) inhibitor Ro 31-8220 (1 x 10(-6) M) significantly reduced this effect. In contrast, the leakage of CC in response to A23187 (1 x 10(-4) M) was not affected by Ro 31-8220. Peanut agglutinin (PNA) and succinyl concanavalin A (SuccCon A), which do not bind to ECs, had no effect at a concentration of 10 micrograms/ml. A lower concentration of WGA (1 microgram/ml) had no significant effect of its own, but significantly reduced the leakage of CC in response to both platelet-activating factor (PAF, 5 x 10(-6) M) and 5-hydroxytryptamine (5-HT, 1 x 10(-4) M), but not to beta-phorbol 12,13-dibutyrate (PDB, 1 x 10(-6) M). These results suggest that all these effects of WGA and Con A involve cell surface receptors, albeit in a non-specific way. A possible mode of action is discussed.

Competitive titration for probing low-abundance ion channel mRNA molecules in normal and regionally-ischaemic heart tissue.

We have measured the expression levels of a range of distinct ion channel genes in the apex/ventricle region and sino-atrial node (SAN) sub-regions of heart under conditions in which conventional Northern hybridization or ribonuclease protection methods were too insensitive or non-quantitative. The abundance of six potassium channel mRNAs was determined in relation to a single synthetic competitor RNA template which was co-reverse transcribed and PCR-amplified. By these methods we have shown that coronary artery ligation procedures which induce anoxia and ischaemic scarring in the apical region reduce amplifiable message abundance in a time-dependent, but non-specific manner. There was no evidence for any selective reduction of individual mRNA levels during this process. Despite a high reproducibility of titration endpoints, competitive RNA template amplification assays did not provide a simple marker for ischaemic damage, since it was not possible to control for tissue sample heterogeneity. We have also applied these competitive nucleic acid titration techniques to demonstrate expression of cAMP- and cGMP-gated ion channel sequences in small pieces of tissue derived from the SAN sub-region of rabbit heart. Although the ion channels encoded by these sequences are obligately coupled to intracellular signalling agonists commonly found in cardiac cells, they have not been described in functional terms within SAN or any other cardiac subregion. For rapid determination of cDNA molecular numbers, we have devised single-gene, DNA template controls to measure absolute abundance of a cAMP-gated cDNA derived from heart tissue. Competitive titration procedures therefore provide an important technique for probing gene induction and/or repression accompanying pharmacological or surgical interventions, or in progression of disease states. For rare cDNAs, they can estimate the representativeness of a given preparation prior to library construction, screening and retrieval of clones, while eliminating 'false positive' or 'false negative' signals.

Possible involvement of microtubules in platelet-activating factor-induced increases in microvascular permeability in vitro.

The blood vessels of the rat small intestine were perfused in vitro with a gelatin-containing physiological salt solution (GPSS). The addition of platelet-activating factor (PAF, 5 microM), podophyllotoxin (50 microM), colcemid (50 microM), or nocodazole (50 microM) to the GPSS for 5 min caused an increase in vascular permeability. This was manifested as an increased trapping of circulating colloidal carbon (CC) within the walls and was assessed using semiautomated image analysis. Pretreatment for 10 min with taxol (5 microM) in the perfusate significantly reduced the permeability-enhancing effects of all four agonists. Since podophyllotoxin, colcemid, and nocodazole are all microtubule-disrupting agents, and since taxol is a microtubule-stabilizing agent, these results suggest that microtubules are involved in the response of the microvessels to PAF. An explanation based on "tensegrity" or "force-counterbalance" is put forward to account for these findings.

Involvement of protein kinase C in the control of microvascular permeability to colloidal carbon.

The vasculature of the rat small intestine and attached mesentery was perfused in vitro with a gelatin-containing physiological salt solution (GPSS). The inclusion of colloidal carbon (CC) in the perfusate towards the end of the experimental period enabled the "leakiness" of microvessels in the villi to be determined, since "leaky" vessels trap CC in their walls. Addition to the perfusate of the inflammatory agonists platelet-activating factor (PAF, 5 x 10(-6) M) or 5-hydroxytryptamine (5-HT, 1 x 10(-4) M), or the microtubule-disrupting agents podophyllotoxin (5 x 10(-5) M), or colcemid (5 x 10(-5) M), or the protein kinase C (PKC) activator phorbol 12, 13-dibutyrate (PDB, 1 x 10(-6) M), caused significantly increased microvascular "blackening" as assessed by image analysis. 4 alpha-phorbol 12, 13-didecanoate (PDD, 1 x 10(-6) M) had no effect. Pretreatment with the PKC inhibitor Ro 31-8220 [corrected] (1 x 10(-6) M) significantly reduced the effects of PAF, 5-HT and PDB, but not those of podophyllotoxin or colcemid. These results suggest, therefore, that PKC is involved in the permeability-enhancing effects of PAF, 5-HT and PDB. Pretreatment with indomethacin (1 x 10(-6) M) as a cyclooxygenase inhibitor did not reduce the response to PDB, indicating that prostaglandin release is of minor importance in the PDB-induced increase in microvascular permeability.

Phorbol 12,13-dibutyrate produces negative inotropism and selective antagonism of responses to adrenoceptor agonists in rat atria.

Rat atria loaded in vitro with the dye INDO-1 produced fluorescence signals indicative of changes in cytoplasmic calcium ion concentration ([Ca2+]c). Such atria showed systolic/diastolic fluctuations indicative of a systolic rise and a diastolic fall in both tension and [Ca2+]c. Positively inotropic responses of the atria to isoprenaline, phenylephrine, ouabain or 4-aminopyridine were associated with fluorescence changes indicative of increased systolic increments in [Ca2+]c. Treatment of atria with phorbol dibutyrate, on the other hand, produced negative inotropism and fluorescence changes indicative of declining systolic increments in [Ca2+]c. Pretreating atria with phorbol dibutyrate diminished both the inotropic and fluorescence responses to subsequent treatment with phenylephrine or isoprenaline, although responses to ouabain or 4-aminopyridine were unchanged. Exposure of atria to a selective inhibitor of protein kinase C mitigated the effects of pretreatment with phorbol dibutyrate, but failed to modify responses to phenylephrine or isoprenaline that were produced in the absence of phorbol dibutyrate.