Effects of catechol ring fluorination on cardiovascular and renal activities of fenoldopam enantiomers

SK&F 87516 is a potent DA₁ receptor agonist with demonstrated renal vasodilator activity. SK&F 87516 is the 6-fluoro analog of another DA₁ agonist/renal vasodilator agent, fenoldopam. SK&F 87516 is a racemic mixture of two enantiomers, SK&F(R)-87516 and SK&F(S)-87516, and like fenoldopam, the (R)-enantiomer is responsible for the biological activities of the racemate. SK&F(R)-87516 is diuretic in spontaneously hypertensive rats and in dogs, whereas its enantiomer, SK&F(S)-87516 is inactive. SK&F(R)-87516 increases glomerular filtration rate, an effect which may account, in part, for its diuretic activity. Unlike fenoldopam, SK&F(R)-87516 is not associated with acute hypotensive activity, tachycardia, or stimulation of the renin-angiotensin-aldosterone system. The activity differences between SK&F(R)-87516 and fenoldopam are not related to differences in DA₁ agonist potency. The activity differences may be due to the differing effects of fluorine and chlorine on the electron distribution in the catechol ring, resulting in an enhanced effect of SK&F(R)-87516 at α₂-adrenoceptors. © 1994 Wiley-Liss, Inc.     

Reversal of mu-opioid-mediated respiratory depression by alpha 2-adrenoceptor antagonism.

The present study was performed in order to evaluate the effects of the selective alpha 2-adrenoceptor antagonist 6-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benzazepine (SK&F 86466) on dermorphin-induced analgesia, respiratory depression and inhibition of locomotor activity in the conscious rat. Intracerebroventricular (icv) administration of dermorphin (3 nmol/rat) decreased respiration rate and relative ventilatory minute volume maximally by 38% and 50% of baseline respectively. SK&F 86466 dose-dependently reversed the dermorphin-induced depression of ventilatory parameters, while SK&F 86466 exerted no effect on dermorphin-induced analgesia or depression of locomotor activity due to catalepsia. It appears, therefore, that alpha 2-adrenoceptors selectively interact with mu 2-opioid-receptor mediated effects, such as respiratory depression, but are not involved in the modulation of mu 1-opioid-related effects, such as supraspinal analgesia and depression of locomotor activity.     

Inhibition of Sterol Biosynthesis and Stem Elongation of Tobacco Seedlings Induced by Some Hypocholesterolemic Agents

Incorporation of DL-[2-¹⁴C]mevalonic acid ([2-¹⁴C]MVA) into4-desmethylsterols in Nicotiana tabacum cv. Turkish Samson seedlingswas inhibited by SK&F 7997-A₃,¹ SK&F 7732-A₃, AY 9944,and the plant growth retardant, Amo 1618. Reductions in 4-desmethylsterol levels resulted from treatmentwith AY 9944 and Amo 1618, but not the SK&F compounds. Amo1618 and SK&F 7997-A₃ both significantly reduced the specificactivity of each of the four major 4-desmethylsterols examined.Although SK&F 7732-A₃ reduced the specific activity of campesterol,and AY 9944 reduced the specific activity of stigmasterol, neitherhad an effect on the specific activity of ß-sitosterol. Stem elongation of tobacco seedlings was retarded by SK&F7997-A₃, AY 9944, and SK&F 7732-A₃, particularly the former,and the retarded plants thus produced were morphologically indistinguishablefrom the Amo 1618-treated plants. Application of exogenous stigmasterol,or GA₃, to the chemically-retarded plants resulted in a reversalof stem growth retardation.     

SK&F 96365, a novel inhibitor of receptor-mediated calcium entry.

A novel inhibitor of receptor-mediated calcium entry (RMCE) is described. SK&F 96365 (1-(beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H- imidazole hydrochloride) is structurally distinct from the known 'calcium antagonists' and shows selectivity in blocking RMCE compared with receptor-mediated internal Ca2+ release. Human platelets, neutrophils and endothelial cells were loaded with the fluorescent Ca2(+)-indicator dyes quin2 or fura-2, in order to measure Ca2+ or Mn2+ entry through RMCE as well as Ca2+ release from internal stores. The IC50 (concn. producing 50% inhibition) for inhibition of RMCE by SK&F 96365 in platelets stimulated with ADP or thrombin was 8.5 microM or 11.7 microM respectively; these concentrations of SK&F 96365 did not affect internal Ca2+ release. Similar effects of SK&F 96365 were observed in suspensions of neutrophils and in single endothelial cells. SK&F 96365 also inhibited agonist-stimulated Mn2+ entry in platelets and neutrophils. The effects of SK&F 96365 were independent of cell type and of agonist, as would be expected for a compound that modulates post-receptor events. Voltage-gated Ca2+ entry in fura-2-loaded GH3 (pituitary) cells and rabbit ear-artery smooth-muscle cells held under voltage-clamp was also inhibited by SK&F 96365; however, the ATP-gated Ca2(+)-permeable channel of rabbit ear-artery smooth-muscle cells was unaffected by SK&F 96365. Thus SK&F 96365 (unlike the 'organic Ca2+ antagonists') shows no selectivity between voltage-gated Ca2+ entry and RMCE, although the lack of effect on ATP-gated channels indicates that it discriminates between different types of RMCE. The effects of SK&F 96365 on functional responses of cells thought to be dependent on Ca2+ entry via RMCE were also studied. Under conditions where platelet aggregation is dependent on stimulated Ca2+ entry via RMCE, the response was blocked by SK&F 96365 with an IC50 of 15.9 microM, which is similar to the IC50 of 8-12 microM observed for inhibition of RMCE. Adhesion and chemotaxis of neutrophils were also inhibited by SK&F 96365. SK&F 96365 is a useful tool to distinguish RMCE from internal Ca2+ release, and to probe the role of RMCE in mediating functional responses of cells. However, SK&F 96365 is not as potent (IC50 around 10 microM) or selective (also inhibits voltage-gated Ca2+ entry) as would be desirable, so caution must be exercised when using this compound.     

Effect of a dopamine D-1 agonist in rats treated chronically with zuclopenthixol

We have previously demonstrated long-lasting increases in vacuous chewing movements (VCM) and tongue protrusions in rats treated discontinuously (DISC), but not continuously (CONT), with neuroleptics. To test whether this increase in mouth movements could be a result of exaggerated activity at the D-1 site, 34 rats were divided into three groups receiving the neuroleptic zuclopenthixol (ZU) DISC or CONT for 15 weeks, or no treatment. After withdrawal DISC treated animals showed an increase in oral activity compared to CONT treated. Two weeks after termination of medication the animals were tested with the D-1 agonist SK&F 38393. The increases in VCM after SK&F 38393 did not differ among the groups, but in contrast to control (CTRL) rats, treated rats showed a significant increase in tongue protrusions. There were no significant differences in the densities of D-1 and D-2 receptors in the striatum between the groups. The increase in tongue protrusions after SK&F 38393 in neuroleptic treated animals implies behavioural D-1 receptor supersensitivity. No significant differences in the rise in tongue protrusions and VCM after SK&F 38393 were seen between DISC and CONT treated animals. Our results thus do not indicate that increased D-1 receptor responsiveness is significant for the rise in spontaneous oral activity found after neuroleptic withdrawal.     

Suppression of EGF-induced cell proliferation by the blockade of Ca2+ mobilization and capacitative Ca2+ entry in mouse mammary epithelial cells

The role of intracellular Ca2+ stores and capacitative Ca2+ entry on EGF-induced cell proliferation was investigated in mouse mammary epithelial cells. We have previously demonstrated that EGF enhances Ca2+ mobilization (release of Ca2+ from intracellular Ca2+ stores) and capacitative Ca2+ entry correlated with cell proliferation in mouse mammary epithelial cells. To confirm their role on EGF-induced cell cycle progression, we studied the effects of 2,5-di-tert-butylhydroquinone (DBHQ), a reversible inhibitor of the Ca2+ pump of intracellular Ca2+ stores, and SK&F 96365, a blocker of capacitative Ca2+ entry, on mitotic activity induced by EGF. Mitotic activity was examined using an antibody to PCNA for immunocytochemistry. SK&F 96365 inhibited capacitative Ca2+ entry in a dose-dependent manner (I50: 1-5 microM). SK&F 96365 also inhibited EGF-induced cell proliferation in the same range of concentration (I50: 1-5 microM). DBHQ suppressed [Ca2+]i response to UTP and thus depleted completely Ca2+ stores at 5 microM. DBHQ also inhibited EGF-induced cell proliferation at an I50 value of approximately 10 microM. The removal of these inhibitors from the culture medium increased the reduced mitotic activity reversibly. Using a fluorescent assay of DNA binding of ethidium bromide, no dead cells were detected in any of the cultures. These results indicate that the inhibitory effects of SK&F 96365 and DBHQ on cell proliferation were due to the inhibition of capacitative Ca2+ entry and Ca2+ mobilization suggesting the importance of capacitative Ca2+ entry and Ca2+ mobilization in the control of EGF-induced cell cycle progression in mouse mammary epithelial cells.     

Mitosis-arresting effect of the calcium channel inhibitor SK&F 96365 on human leukemia cells.

The effect of SK&F 96365 (1-(beta-[3-(4-methoxyphenyl)propoxyl]-4- methoxyphenethyl)-1H-imidazole hydrochloride), a recently synthesized inhibitor of receptor-mediated calcium entry, was investigated on human hematopoietic cell lines. We found that treatment of the T-cell leukemia line Jurkat with SK&F 96365 inhibited the Ca2+ influx triggered by antibodies against the CD3/TCR complex, while the inositol trisphosphate-dependent Ca2+ release from intracellular stores remained intact. A 50% inhibition of the Ca2+ influx was obtained with 5 microM SK&F 96365, while higher concentrations of the drug blocked the CD3-dependent Ca2+ influx completely. In addition to its blocking of the Ca2+ influx, treatment with SK&F 96365 was found to accumulate mitotic cells. The drug (5 microM) imposed a total cell cycle arrest in G2/M. The mitosis block could be reversed by removal of the inhibitor from the cultures, while elevation of intracellular or extracellular Ca2+ did not restore cell cycle progression. This suggests that the cell cycle block induced by SK&F 96365 is not directly related to its action as an inhibitor of receptor-mediated calcium entry. Our findings indicate that SK&F 96365, in addition to its ability to inhibit receptor-triggered Ca2+ influx, offers a new method for imposing a reversible mitosis arrest in hematopoietic cell lines.     

Characterization of the hemodynamic activities of fenoldopam and its enantiomers in the dog

Fenoldopam (SK&F 82526) is a potent and selective dopamine DA-1 agonist with demonstrated renal vasodilator and antihypertensive activities in experimental animals and humans. Fenoldopam is a racemic mixture of two enantiomers, SK&F R-82526 and SK&F S-82526. The R-enantiomer is uniformly reported to be more potent than the racemate; in contrast, there is controversy regarding potency of the S-enantiomer. In these studies, the renal and systemic hemodynamic activities of fenoldopam and its enantiomers are characterized in anesthetized, phenoxybenzamine-treated dogs. The results show that the renal and systemic vasodilator activities of fenoldopam are properties of the R-enantiomer; the S-enantiomer is essentially inactive. The renal and systemic vasodilator properties of SK&F R-82526 are antagonized in a competitive fashion by the DA-1 antagonist, SK&F R-83566, but not the DA-2 antagonist, domperidone. Ganglionic blockade did not attenuate renal vasodilation associated with SK&F R-82526. Thus, the mechanism of SK&F R-82526-associated vasodilation, like that previously established for fenoldopam, is via stimulation of postganglionic DA-1 receptors.     

The discriminative stimulus properties of the D-1 agonist SK&F 38393 and the D-2 agonist (-)-NPA are mediated by separate mechanisms

The dopamine D-1 agonist SK&F 38393 (10 mg/kg) the D-2 agonist (-)-NPA (0.04 mg/kg) and d-amphetamine (1.0 mg/kg) were established as discriminative stimuli versus saline in rats. The stimulus induced by SK&F 38393 was stereoselective, since the R-(+)-, but not the S-(-)-enantiomer was effective. It was mimicked by two partial D-1 agonists with central effects, SK&F 75670 and Lu 24-040, but not by the peripheral agonist fenoldopam. D-2 agonists and d-amphetamine were ineffective. The effect of SK&F 38393 was antagonized by SCH 23390, but not by its inactive enantiomer SCH 23388 or by the D-2 antagonist YM 09151-2. The (-)-NPA stimulus was dependent on postsynaptic D-2 receptors: It was mimicked by quinpirole and pergolide in stimulant dosages, whereas the partial D-2 agonist (-)-3-PPP inhibited the effect of (-)-NPA. The dopamine synthesis inhibitor alpha-methyl-p-tyrosine did not antagonize the effect of (-)-NPA. Likewise, the above-mentioned D-1 agonists produced saline responding. D-amphetamine produced partial substitution to (-)-NPA. The (-)-NPA stimulus was blocked by YM 09151-2, but not by SCH 23390. In d-amphetamine-trained rats, quinpirole, (-)-NPA and pergolide produced generalization, whereas SK&F 38393 was ineffective. Both SCH 23390 and YM 09151-2 antagonized the effect of d-amphetamine. It is concluded that the cues induced by SK&F 38393 and (-)-NPA are mediated by separate D-1 and D-2 sites, whereas both sites contribute to the effect of d-amphetamine.     

Effects of the thromboxane receptor antagonist SK&F 88046 in the canine, monkey and human coronary vasculature

U-46619, a stable "functional" thromboxane/endoperoxide receptor agonist, produced potent contractile responses in isolated canine, rhesus monkey and human left circumflex coronary arteries (EC50 = 9.11 x 10(-9)M, 1.98 x 10(-8)M and 3.50 x 10(-9)M, respectively). Canine intrapulmonary veins were also contracted potently by U-46619 (EC50 = 1.22 x 10(-9)M). SK&F 88046, a thromboxane A2 (TxA2) end-organ receptor antagonist, blocked the vasoconstrictor effects of U-46619 in the canine circumflex artery (KB = 1.33 x 10(-8)M), canine intrapulmonary vein (KB = 1.46 x 10(-9)M), monkey circumflex artery (KB = 8.47 x 10(-8)M), and human circumflex artery (KB = 8.49 x 10(-7)M). SK&F 88046 was 10-60 times more potent in the canine and rhesus monkey coronary vasculature than in the human coronary preparations. Intracoronary administration of U-46619 to anesthetized, open chest dogs produced a dose-related decrease in left circumflex coronary artery blood flow which resulted in decreases in left ventricular developed pressure, left ventricular positive and negative dP/dt, ascending aortic blood flow, and an increase in left ventricular end-diastolic pressure. The decrease in coronary blood flow and the hemodynamic changes were either attenuated or completely inhibited by i.v. administration of SK&F 88046 (2.5 mg/kg + 0.05 mg/kg/min or 5.0 mg/kg + 0.1 mg/kg/min). SK&F 88046 was compared to two other TxA2 receptor antagonists in canine isolated intrapulmonary veins. SQ 29,548 was approximately 2-times more potent than SK&F 88046 as an antagonist of U-44619 mediated contractions (KB = 7.0 x 10(-10)M). In contrast, BM 13.177 was 150-fold less potent (KB = 2.19 x 10(-7)M) than SK&F 88046. Thus, the present study demonstrates species variability in response to TxA2 agonists and antagonists and reconfirms the relative importance of species selection in studying these agents.     

Salt-induced hypertension in chronic renal failure: Evidence for a neurogenic mechanism

We investigated the possibility that blood pressure elevation induced by salt excess may be secondary to a neurogenic mechanism. The compound SK&F 64139 (50 mg/kg) known to inhibit central and peripheral phenylethanolamine N-methytransferase (PNMT) the enzyme necessary for the conversion of norepinephrine to epinephrine, was given by oral gavage to two groups of subtotally nephrectomized rats maintained for five days on either a high salt (HS) or low salt (LS) diet respectively. Blood urea nitrogen (BUN) and hematocrit were not different between the two groups, while body weight and serum Na were significantly higher in the HS animals. Baseline mean blood pressure (BP) was higher in the HS animals (HS 154 ± 4.7 vs LS 121 ± 3.7 mmHg, p<0.001) and decreased by 39 ± 6.9 mmHg one and one half hour post SK&F 64139 to normotensive levels in the HS as opposed to a decrease of 10 ± 1.8 mmHg in the LS group. Baseline heart rate (HR) was higher in the LS group (474 ± 17 beats/min) vs the HS group (408 ± 17, p<0.05), and decreased significantly after SK&F 64139 in both groups to the same extent (by 17.6% in the HS vs 13.3% in the LS). A third group of subtotally nephrectomized rats maintained for five days on a HS diet were given by oral gavage the compount SK&F 29661 (100 mg/kg), a PNMT inhibitor which does not cross the blood-brain barrier. Following SK&F 29661, there was no significant decrease in mean BP (153 ± 5 to 149 ± 4 mmHg) and a less than 2% decrease in HR. Baseline plasma norepinephrine (NE) was higher in the HS as compared to the LS group (1.50 ± 0.16 vs 0.904±0.15 ng/ml, respectively, p<0.05) and a significant correlation was found between plasma NE level and decrease in BP following SK&F 64139 (r=0.65, p<0.01). Not withstanding possible effects of some ancillary properties of SK&F 64139, these data support the hypothesis that a neurogenic component, possibly mediated via central epinephrine containing neurons, contributes to the BP elevation induced by salt excess.     

Selective antagonism of peptidoleukotriene responses does not reduce myocardial damage or neutrophil accumulation following coronary artery occlusion with reperfusion

This study was designed to assess the effect of a peptidoleukotriene receptor antagonist, SK&F 104353, for limiting myocardial damage and neutrophil accumulation in rats subjected to myocardial reperfusion injury (MI/R). In conscious rats, SK&F 10,4353 (25 mg/kg, i.v.) antagonized LTD4-induced vasopressor responses by 90% and 60% at 1 and 4 hr, respectively, indicating effective blockade of peptido-leukotriene responses. In another group of animals subjected to 30 min of coronary artery occlusion with reperfusion for 24 hr, myocardial injury and neutrophil infiltration were determined by measuring creatine phosphokinase (CPK) specific activity and myeloperoxidase (MPO) activity, respectively, in the left ventricular free wall (LVFW). Myocardial CPK levels were 8.1 +/- 0.2 U/mg protein in Sham-MI/R vehicle-treated animals, and were significantly decreased to 6.4 +/- 0.6 U/mg protein in MI/R-vehicle animals. Myocardial MPO values were 1.5 +/- 0.5 U/g LVFW in Sham-MI/R vehicle-treated animals, and significantly increased to 4.3 +/- 0.6 U/g LVFW in MI/R-vehicle animals. Administration of SK&F 10,4353 (25 mg/kg, i.v.) 1 min prior to coronary occlusion and 3.5 hr post reperfusion had no effect on the loss of myocardial CPK specific activity or the increase in MPO levels (p greater than 0.05, compared to the MI/R-vehicle group). Thus, at a dose that antagonized LTD4-induced vasopressor responses, SK&F 104353 did not attenuate either the extent of myocardial injury or inflammatory cell accumulation associated with myocardial ischemia/reperfusion. These results suggest that peptidoleukotrienes do not contribute to the progression of myocardial ischemic/reperfusion injury.     

SK&F 96365 inhibits intracellular Ca pumps and raises cytosolic Ca concentration without production of nitric oxide and von Willebrand factor

The effects of the imidazole compound SK&F 96365 on Ca²⁺ movements and production of nitric oxide (NO) and von Willebrand factor (vWF) have been investigated in human endothelial cells. Changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) were measured with Fura-2. Real-time production of NO was monitored with a porphyrinic microsensor and the release of vWF with an enzyme-linked immunosorbent assay. Irrespective of the transmembrane Ca²⁺ gradient, 30 μM SK&F 96365 doubled [Ca²⁺]_i suggesting a Ca²⁺ release from intracellular stores. The SK&F 96365-induced [Ca²⁺]_i rise was not accompanied by detectable NO and vWF production, while 1 μM thapsigargin enhanced [Ca²⁺]_i 2.5 times, doubled the secretion of vWF and increased the NO production to 10 ± 4 nM (n = 5). Pretreatment with SK&F 96365 prevented thapsigargin from increasing [Ca²⁺]_i, NO production and vWF secretion. To investigate the mechanism by which SK&F 96365 released Ca²⁺, from internal pools, its effect and that of thapsigargin on the ATP-dependent ⁴⁵Ca²⁺, uptake into platelet membrane vesicles were compared. SK&F 96365 as thapsigargin, dose-dependently reduced the initial rate of ⁴⁵Ca²⁺ uptake. In conclusion, we demonstrate that, in the absence of Ca²⁺ entry from the extracellular space, the [Ca²⁺]_i increase elicited by SK&F 96365 or thapsigargin is not sufficient to initiate NO synthesis and vWF secretion. This confirms the important role of Ca²⁺ influx in endothelial secretion processes.     

Selective antagonism of peptidoleukotriene response does not reduce myocardial damage opr neutrophil accumulation following coronary artery occlusion with reperfusion

This study was designed to assess the effect of a peptidol eukotriene receptor antagonist. SK&F 104353, for limiting myocardial damage and neutrophil accumulation in rats subjected to myocardial reperfusion injury (MI/R). In conscious rats, SK&F 104353 (25 mg/kg, i.v.) antagonized LTD₄-induced vasopressor responses by 90% and 60% at 1 and 4 hr, respectively, indicating effective blockade of peptidol eukotriene responses. In another group of animals subjected to 30 min of coronary artery occlusion with reperfusion for 24 hr, myocardial injury and neutrophil infiltration were determined by measuring creatine phosphokinase (CPK) specific activity and myeloperoxidase (MPO) activity, respectively, in the left ventricular free wall (LVFW). Myocardial CPK levels were 8.1 ± 0.2 U/mg protein in Sham-MI/R vehicle-treated animals, and were significantly decreased to 6.4 ± 0.6 U/mg protein in MI/R-vehicle animals. Myocardial MPO values were 1.5 ± 0.5 U/g LVFW in Sham-MI/R vehicle-treated animals, and significantly increased to 4.3 ± 0.6 U/g LVFW in MI/R-vehicle animals. Administration of SK&F 105353 (25 mg/kg, i.v.) 1 min prior to coronary occlusion and 3.5 hr post reperfusion and no effect on the loss of myocardial CPK specific activity or the increase in MPO levels (p > 0.05, compared to the MI/R-vehicle group). Thus, at a dose that antagonized LTD₄-induced vasopressor responses, SK&F 104353 did not attenuate either the extent of myocardial injury or inflammatory cell accumulation associated with myocardial ischemia/ reperfusion. These results suggest that peptidoleukotrienes do not contribute to the progression of myocardial ischemic/reperfusion injury.     

Calmodulin discriminates between the two enantiomers of the receptor-operated calcium channel blocker SK&F 96365: a study using 1H-NMR and chiral HPLC

1H nuclear magnetic resonance at 360 MHz shows that SK&F 96365 (1-(beta-[3-(p-methoxyphenyl)-propyloxy]-p-methoxyphenethyl)-1H- imidazole hydrochloride), an antagonist of mammalian receptor-operated calcium channels, interacts with the calcium-binding regulatory protein calmodulin (CaM). This may be inferred by a number of chemical shift changes in the spectrum of the calcium-saturated protein induced by addition of the compound. Moreover, two well-resolved singlets corresponding to the 2-proton of the SK&F 96365 imidazolium moiety are observed in the spectrum over a wide range of protein:compound ratios. Separation of rac SK&F 96365 into its two enantiomers by high-performance liquid chromatography on a cellulose tris (4-methylbenzoate) column enabled us to show that the doubling of this NMR signal in the presence of CaM is due to a propensity of the protein to distinguish between the two optical isomers of the compound.     

Inhibition of the sodium channel by SK&F 96365, an inhibitor of the receptor-operated calcium channel,in mouse diaphragm

The effects of SK&F 96365, a blocker of the receptor-operated Ca²⁺ channel, on contractilities and the Na⁺ channel of mouse diaphragm were studied. SK&F 96365 (10–50 µM) reversibly inhibited twitches, tetanic contractions and muscle and nerve action potentials. The IC₅₀ was 17–24 µM. The inward Na⁺ current was suppressed and its recovery from inactivations delayed. Crotamine, a peptide toxin that binds to neurotoxin receptor site 3 of the muscle Na⁺ channel, enhanced the inhibitory effects of SK&F 96365 and reduced the IC₅₀ to about 4 µM. Veratridine had similar effects, although it was less effective than crotamine. On the other hand, the crotamine-induced membrane depolarizations and spontaneous discharges of muscle action potentials were inhibited by SK&F 96365 noncompetitively. The inhibitory effects of tetrodotoxin and tetracaine were additive with those of SK&F 96365 but were enhanced slightly by crotamine. The results suggested that SK&F 96365 acts on a distinct site and blocks the Na⁺ channel of excitable membranes at a concentration range that inhibits the receptor-operated calcium channel.     

Renal effects of SK&F 82526 in anesthetized rats

Dopamine affects renal hemodynamics, renal tubular functions, and the secretion of renin. We have studied the renal effects of SK&F 82526 (an agonist which is selective for the DA1 subclass of dopamine receptors) in anesthetized rats. Infused intravenously at 0.005 mumol/min/kg, this drug increased renal plasma flow and the clearances of PAH and insulin, effects which are consistent with decreased renovascular resistance. Concomitantly, urine flow and K excretion increased, and Na excretion tended to increase. All these effects of SK&F 82526 were antagonized by intravenous metoclopramide (1 mumol/min/kg). Despite its diuretic effect and despite its lack of effect on arterial blood pressure, SK&F 82526 increased arterial plasma renin concentration, suggesting a stimulatory effect on renin secretory rate. Taken together, our results demonstrate that the renal effects of SK&F 82526 mimic those of dopamine.     

Metabolic consequences of positive inotropy in rat and guinea-pig hearts

The metabolic and physiological responses of hearts from male rats and guinea-pigs to isoprenaline and SK&F 94120 (a phosphodiesterase III inhibitor), have been studied. Doses which gave similar chronotropic stimulation gave different inotropic responses. In both species, isoprenaline generated a greater increase in developed tension than SK&F 94120. With both drugs the inotropic response in the rat was less than than in the guinea-pig. 31P-NMR investigation of high-energy phosphate levels showed reduction in PCr concentration and an accompanying acidosis in the isoprenaline-perfused rat heart only. In both species, lactate production was stimulated by SK&F 94120 but not by isoprenaline. These results are discussed with reference to G-protein activation of ion channels and differences in Ca2+ handling by the two species.     

Preliminary identification and role of phosphodiesterase isozymes in human basophils.

We attempted to identify and establish the role of cyclic nucleotide phosphodiesterase (PDE) isozymes in human basophils by using standard biochemical techniques as well as describing the effects of isozyme-selective and nonselective inhibitors of PDE. The nonselective PDE inhibitors, theophylline and 3-isobutyl-1-methylxanthine, inhibited anti-IgE-induced release of histamine and leukotriene C4 (LTC4) from basophils. This inhibition was accompanied by elevations in cAMP levels. Rolipram, an inhibitor of the low Km cAMP-specific PDE (PDE IV), inhibited the release of both histamine and LTC4 from activated basophils and increased cAMP levels in these cells. In contrast, mediator release from basophils was not inhibited by either siguazodan or SK&F 95654, inhibitors of the cGMP-inhibited PDE (PDE III) or zaprinast, an inhibitor of the cGMP-specific PDE (PDE V). SK&F 95654 failed to elevate basophil cAMP in these experiments whereas zaprinast induced significant increases in cAMP content. The inhibitory effect of rolipram on mediator release was potentiated by siguazodan or SK&F 95654, but not by zaprinast. SK&F 95654 also enhanced the ability of rolipram to increase cAMP content. Forskolin, a direct activator of adenylate cyclase, inhibited IgE-dependent release of mediators from basophils and increased cAMP levels in these cells. These effects were enhanced by rolipram, but not by SK&F 95654 or zaprinast. The cell permeant analog of cAMP, dibutyryl cAMP, inhibited mediator release from these cells, a property not shared by either dibutyryl-cGMP or sodium nitroprusside, an activator of soluble guanylate cyclase. The presence of both PDE III and PDE IV was confirmed by partially purifying and characterizing PDE activity in broken cell preparations. Overall, these data lend support to the hypothesis that cAMP inhibits mediator release from basophils and suggest that the major PDE isozyme responsible for regulating cyclic AMP content in these cells is PDE IV, with a minor contribution from PDE III. However, the finding that zaprinast caused increases in cAMP without inhibiting mediator release indicates that cAMP accumulation is not invariably linked to an inhibition of basophil activation.     

Spectroscopic and physicochemical studies on the interactions of reversible H+/K(+)-ATPase inhibitors with phospholipid bilayers

Three complementary techniques, differential scanning calorimetry (DSC), Fourier transform infrared (FT-IR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, have been used to characterise the interactions between dimyristoylphosphatidylcholine (DMPC) model biological membranes and two non-covalent inhibitors of the gastric (H+, K+)-ATPase. DSC, FT-IR and deuterium NMR studies of side-chain perdeuterated DMPC (DMPC-d54) support the prediction, based on physical property measurements, that SK&F 96079 partitions readily into phospholipid bilayers, resulting in a slight but measurable disordering of the lipid hydrocarbon side-chain motion and a concomitant reduction in the co-operativity and onset temperature of the gel to liquid crystalline phase transition. However, FT-IR and deuterium NMR studies show that the bilayer structure remains intact even at high (1:4) compound to lipid molar ratios. Proton (1H) NMR nuclear Overhauser effect determinations in sonicated codispersions reveal details of the membrane bound conformations of SK&F 96079. The structurally related analogue SK&F 96464, also studied by 1H-NMR, can be shown, by interpreting the effects of nitroxide-labelled fatty acid relaxation probes, to adopt a well-defined orientation relative to the bilayer, in contrast to SK&F 96079. This orientation directs the proton at the 5-position of the quinoline ring towards the hydrophobic centre of the bilayer, and the quinoline 8-methoxy group towards the surface and hence the aqueous phase. Molecular modelling has been used to rationalise this orientation in terms of hydrogen bonds between the amino NH group of SK&F 96464 and the sn-1 carbonyl group of DMPC, and between the NH group of the protonated quinoline ring of SK&F 96464 and the DMPC phosphodiester group.