Effect of neuromediating and neuroblockading hormones on the RNA synthesis in the rat liver nucleus

It was shown that rRNA and HnRNA synthesis in rat liver nuclei does not change-within 30 min after intraperitoneal injection of acetylcholine (0.005 mg per 100 g of body weight) but decreases after injection of norepinephrine and epinephrine (0.05 mg per 100 g of body weight). The synthesis of rRNA (but not of HnRNA) increases after injection of hydrocortisone (2,5 mg per 100 g of body weight). The synthesis of HnRNA (but not of rRNA) increases after injection of ACTH1-24 (3 ME per 100 g of body weight) and oxytocin (1 ME per 100 g of body weight). The synthesis of rRNA decreases after injection of propranolol and atropine (0.5 mg per 100 g of body weight). At the same time, the synthesis of HnRNA does not change thereby. The inhibitory effect of propranolol and atropine was corrected by electrostimulation of hypothalamus. The content of cAMP and Ca2+ and the phosphorylation degree of nuclear proteins are increased after stimulation of hypothalamus. The phosphorylation of nuclear proteins is increased by 10(-8)-10(-6) M cAMP. The synthesis of RNA in liver nuclei is increased by 10(-6) M cAMP only after addition of cytosol. In this case the activity of RNA-polymerase II increases in a greater degree than that of RNA-polymerase I + III. It is assumed that the regulatory mechanisms of rRNA and HnRNA synthesis are different. The role of hypothalamus electrostimulation, neurotransmitters, hormones, and cAMP in the mechanisms of RNA synthesis in rat liver nuclei is discussed.     

Phospholipid hydroperoxide accumulation in liver of rats intoxicated with carbon tetrachloride and its inhibition by dietary alpha-tocopherol

The formation and accumulation of phospholipid hydroperoxides, especially of phosphatidylcholine hydroperoxide (PCOOH), a primary peroxidation product of phosphatidylcholine (PC), in livers of carbon tetrachloride-intoxicated rats was investigated. PCOOH in liver and blood plasma was measured by a chemiluminescence-high-performance liquid chromatography procedure originally developed by Miyazawa et al. (Anal. Lett. 20, 915, 1987; Free Radical Biol. Med. 7, 209, 1989). Male Sprague-Dawley rats (120 g body wt., 5 weeks of age) were used in the experiments. The amount of PCOOH in the liver of control rats (CCl4-untreated) was 160 +/- 20 pmol/100 mg protein (mean +/- SD) and the PCOOH/PC molar ratio was 1.1 +/- 0.1 X 10(-5). In CCl4 (0.1 ml/100 g body wt.)-dosed rats, the liver PCOOH was 289 +/- 65 pmol/100 mg protein (PCOOH/PC = 2.4 +/- 0.4 X 10(-5], 764 +/- 271 pmol/100 mg protein (PCOOH/PC = 5.2 +/- 1.7 X 10(-5], and 856 +/- 165 pmol/100 mg protien (PCOOH/PC = 6.0 +/- 0.8 X 10(-5] at 6 h, 24 h, and 1 week after the dose, respectively. Under such conditions, the liver phosphatidylethanolamine hydroperoxide (PEOOH) level was not altered and the concentration was less than 100 pmol/100 mg protein even after the dose. The increments of liver PCOOH were suppressed 56% by the oral supplementation of DL-alpha-tocopherol (5 mg/100 g body wt./day) for a week before CCl4 administration. A relatively larger amount of PEOOH was found after stimulation of PC hydroperoxidation in the liver of rats with a large amount of CCl4 (0.25 ml/100 g body wt.) rather than with the small amount of CCl4 (0.1 ml/100 g body wt.).(ABSTRACT TRUNCATED AT 250 WORDS)     

cAMP-independent casein kinase mediates phosphorylation of many T3-dependent phosphoproteins in rat liver cytosol

Administration of T3 (20 micrograms/100 g BW) for 3 days increases phosphorylation of several proteins in rat liver cytosol in vitro. To help elucidate the mechanism of T3-induced phosphorylation, we studied which protein kinase(s) mediate phosphorylation of endogenous cytosolic proteins. Five different protein kinases were obtained by DEAE+ cellulose column chromatographic fractionation of liver cytosol. When their ability to phosphorylate heat-inactivated cytosol was investigated, casein kinase, a cAMP independent protein kinase, showed the strongest effect. Casein kinase, purified by phosphocellulose chromatography, phosphorylated more than 10 cytosolic proteins. Several T3-dependent (and cAMP independent) phosphoproteins were included among these. One protein with Mr 39 X 10(3), of which phosphorylation is stimulated by T3 within five hours after injection, was the most active substrate for casein kinase. The results suggest that casein kinase is the enzyme responsible for phosphorylation of many rat liver cytosolic proteins and that several phosphoproteins, apparently under T3-regulation, might be phosphorylated by this enzyme.     

In vivo metabolism of 3H-testosterone in adult male rats: effects of estrogen administration.

The metabolism of testosterone (T) was studied in normal adult male rats using a constant infusion of trace amounts of the 3H-steroid into a tail vein for 3 h in order to attain a state of equilibrium. Samples of plasma, liver, kidney, prostate, seminal vesicles and muscle were analysed for 3H-testosterone, 3H-5alpha-dihydrotestosterone (5alphaDHT) and 3H-5alpha-androstanediol (Adiol). When compared to the 3H-T level in plasma there were high levels of 3H-T in kidney and of 3H-5alphaDHT in prostate and seminal vesicles. Intraperitoneal estradiol valerate administration (100 mug/day) for 4 days decreased and 3H-5alphaDHT levels in the prostate and seminal vesicles. The estrogen administration increased the T metabolic clearance rate from 17.5 1/24 h/100 g body wt to 22.6 1/24 h/100 g body wt.     

Acute in vivo stimulation of low-Km cyclic AMP phosphodiesterase activity by insulin in rat-liver Golgi fractions

A low-Km phosphodiesterase activity, which is acutely stimulated by insulin in vivo, has been identified in plasma membranes and Golgi fractions prepared from rat liver homogenates in isotonic sucrose. Within seconds after insulin injection (25 micrograms/100 g body weight) cAMP phosphodiesterase activity increases by 30-60% in Golgi fractions and by 25% in plasma membranes; activity in crude particulate and microsomal fractions is unaffected. The increase in activity is short-lived in the light and intermediate Golgi fractions, but persists for at least 10 min in the heavy Golgi fraction. It precedes the translocation of insulin and insulin receptors to these fractions, which is maximal at 5 min. The doses of insulin required for half-maximal and maximal activation are, respectively, 7.5 micrograms/100 g and 25 micrograms/100 g body weight. Golgi-associated cAMP phosphodiesterase activity shows non-linear kinetics; a high-affinity component (Vmax, 13 pmol min-1 mg protein-1; Km, 0.35 microM) is detectable. Insulin treatment increases the Vmax 60-70%, but does not affect the Km. Unlike the low-Km cAMP phosphodiesterase associated with crude particulate fractions, the Golgi-associated activity is not easily extractable by solutions of low or high ionic strength. On analytical sucrose density gradients, low-Km cAMP phosphodiesterase associated with the total particulate fraction equilibrates at lower densities than endoplasmic reticulum and lysosomal markers, but at a higher densities than plasma membrane, Golgi markers and insulin receptors. Insulin treatment increases the specific activity of the enzyme by 20-60% at densities below 1.12 g cm-3, and by 20-40% in the density interval 1.23-1.25 g cm-3. Such treatment also causes a slight, but significant shift in the distribution of phosphodiesterase towards lower densities. It is suggested that Golgi elements or physically similar subcellular structures are a major site of localization of insulin-sensitive cAMP phosphodiesterase in rat liver. However, internalization of the insulin-receptor complex is probably not required for enzyme activation.     

Hypohydration does not impair skeletal muscle glycogen resynthesis after exercise

The purpose of this investigation was to examine the effects of moderate hypohydration (HY) on skeletal muscle glycogen resynthesis after exhaustive exercise. On two occasions, eight males completed 2 h of intermittent cycle ergometer exercise (4 bouts of 17 min at 60% and 3 min at 80% of maximal O2 consumption/10 min rest) to reduce muscle glycogen concentrations (control values 711 +/- 41 mumol/g dry wt). During one trial, cycle exercise was followed by several hours of light upper body exercise in the heat without fluid replacement to induce HY (-5% body wt); in the second trial, sufficient water was ingested during the upper body exercise and heat exposure to maintain euhydration (EU). In both trials, 400 g of carbohydrate were ingested at the completion of exercise and followed by 15 h of rest while the desired hydration level was maintained. Muscle biopsy samples were obtained from the vastus lateralis immediately after intermittent cycle exercise (T1) and after 15 h of rest (T2). During the HY trial, the muscle water content was lower (P less than 0.05) at T1 and T2 (288 +/- 9 and 265 +/- 5 ml/100 g dry wt, respectively; NS) than during EU (313 +/- 8 and 301 +/- 4 ml/100 g dry wt, respectively; NS). Muscle glycogen concentration was not significantly different during EU and HY at T1 (200 +/- 35 vs. 251 +/- 50 mumol/g dry wt) or T2 (452 +/- 34 vs. 491 +/- 35 mumol/g dry wt). These data indicate that, despite reduced water content during the first 15 h after heavy exercise, skeletal muscle glycogen resynthesis is not impaired.     

Effects of vitamin E, ascorbic acid and mannitol on alloxan-induced lipid peroxidation in rats

ω6- and ω3-unsaturated lipid hydroperoxides decompose to yield pentane and ethane, respectively. Alloxan toxicity was studied in rats in relation to pentane and ethane produced during lipid peroxidation induced by intraperitoneal injection of 20 mg of alloxan/100 g body wt. Fifteen minutes after injection, vitamin E-deficient rats exhaled 102- and 11.2-fold more pentane and ethane, respectively, than prior to injection. Injection of 75 mg ascorbic acid/100 g body wt 30 min prior to alloxan treatment prolonged the time over which peroxidation occurred and all vitamin E-deficient rats died before 4 h. Vitamin E-deficient rats injected with 100 mg of the radical scavenger mannitol/ 100 g body wt 30 min prior to alloxan treatment were completely protected against lipid peroxidation, and none of the rats died by 4 h. Rats fed 40 iu dl-α-tocopherol acetate/kg diet or injected with 100 mg dl-α-tocopherol/100 g body wt were either totally protected against alloxan and alloxan-ascorbic acid-induced peroxidation or were only slightly affected as shown by very low-level pentane and ethane production. Thiobarbituric acid reactants in plasma, liver and pancreas 4 h after alloxan treatment reflected the prooxidant nature of ascorbic acid and alloxan, the vitamin E status of the rats and the protective effect of mannitol. Plasma glucose levels 4 h after alloxan injection were lowest in vitamin E-injected rats and highest in vitamin E-deficient rats. Only in vitamin E-deficient rats were both lipid peroxidation and significantly elevated plasma glucose levels observed by 4 h post-alloxan treatment.     

Cyclic nucleotides levels in the liver of rats treated with CCl4.

Treatment of rats with two different doses of CCl4 (respectively 2.5 and 0.5 ml/kg body wt. intragastrically) is followed by a rapid increase in the cAMP content of the liver. With 0.5 ml of CCl4, the increase occurs as early as 30 min after poisoning, namely about 4-5 h before the onset of triglyceride accumulation in the liver. The maximum increase has been at 6 h after administration of the hepatotoxin. In both experimental conditions, normal values are recovered only after 36-48 h. cGMP level appears unmodified during the whole observation period. Therefore the ratio cGMP/CAMP decreases consistently. The ATP level decreases significantly between 2 and 12 h. The increase in liver triglycerides level after CCl4 can be also a consequence of an impairment of microtubule function, leading to a decreased release of lipoprotein micelles from hepatocytes into the blood stream.     

Cyclid 3':5'-nucleotide phosphodiesterase. Interconvertible multiple forms and their effects on enzyme activity and kinetics.

An extract of rat liver or human platelet displayed three cyclic 3':5'-nucleotide phosphodiesterase activity peaks (I, II, and III) in a continuous sucrose density gradient when assayed with millimolar adenosine 3':5'-monophosphate (cAMP) or guanosine 3':5'-monophosphate (cGMP). The three fractions obtained from each nucleotide were not superimposable. The molecular weights corresponding to the three activity peaks of cAMP phosphodiesterase in rat liver were approximately: I, 22,000; II, 75,000; and III, 140,000. In both tissues, fraction I was barely detectable when assayed with micromolar concentrations of either nucleotide, presumably because fraction I has low affinity for cAMP and cGMP. Any one of the three forms upon recentrifugation on the gradient generated the others, indicating that they were interconvertible. The multiple forms appear to represent different aggregated states of the enzyme. The ratio of the three forms of cAMP phosphodiesterase in the platelet was shifted by dibutyryl cAMP (B2cAMP) and by the enzyme concentration. B2cAMP enhanced the formation of fraction I. Low enzyme concentration favored the equilibrium towards fraction I, while high enzyme concentration favored fraction III. When phosphodiesterase activities in the extract of rat liver, human platelets, or bovine brain were examined as a function of enzyme concentration, rectilinear rates were observed with micromolar, but not with millimolar cAMP or cGMP. The specific activity with millimolar cAMP was higher with low than with high protein concentrations, suggesting that the dissociated form catalyzed the hydrolysis of cAMP faster than that of the associated form. In contrast, the specific activity with millimolar cGMP was lower with low than with high protein concentrations. Supplementing the reaction mixture with bovine serum albumin to a final constant protein concentration did not affect the activity, suggesting that the concentration of the enzyme rather than that of extraneous proteins affected the enzyme activity. A change in enzyme concentration affected the kinetic properties of phosphodiesterase. A low enzyme concentration of cAMP phosphodiesterase yielded a linear Lineweaver-Burk plot, and a Km of 1.2 X 10(-4) M (bovine), 3 X 10(-5) M (platelet), or 5 X 10(-4) M (liver), while a high enzyme concentration yielded a nonlinear plot, and apparent Km values of 1.4 X 10(-4) M and 2 X 10(-5) M (brain), 4 X 10(-5) M and 3 X 10(-6) M (platelet), or 4 X 10(-5) M and 3 X 10(-6) (liver). Since a low enzyme concentration favored fraction I, the dissociated form, whereas a high enzyme concentration favored fraction III, the associated form, these kinetic constants suggest that the dissociated form exhibits a high Km and the associated form exhibits a low Km. In contrast, a high enzyme concentration gave a linear kinetic plot for cGMP phosphodiesterase, while a low enzyme concentration gave a nonlinear plot...     

Rate of release of hepatic triacylglycerol into serum in the starved rat.

After an intravenous injection of a pulse of [U-14C]palmitate to starved rats, the time-dependent radioactivity profiles were determined in the triacylglycerol (triglyceride) of hepatic microsomal fractions, floating fat, mitochondria and nuclei. The profile of activity in serum gave a value of 0.08 mg/min per 100 g body wt. for the irreversible disposal rate of triacylglycerol from serum. This value, combined with the previously estimated rate of movement of triacylglycerol from serum to liver, and the reported rate from intestine to serum, gave a calculated value of 0.35 mg/min per 100 g body wt. for release rate of triacylglycerol from liver to serum. The rate of release of hepatic triacylglycerol into serum was also measured by the widely used Triton WR-1339 method. The rate obtained with this technique (0.15 mg of triacylglycerol/min per 100 g body wt.) was identical with that reported previously. During the interval from 45 min to 3h after ethanol administration this rate increased to 0.18 mg/min per 100 g body wt. It was concluded that the use of Triton underestimates the true rate of movement of triacylglyerol from liver to serum.     

Beneficial effect of somatostatin on galactosamine induced liver injury

The purpose of our investigation was to study the effect of somatostatin on acute experimental liver injury induced in rats by galactosamine (1.2 g/100 g body wt.). Somatostatin (125 micrograms/100 g body wt.) was administered subcutaneously in a protamine sulphate/ZnCl2 suspension either 2 h prior to the injection of galactosamine or 2 h and again 12 h following the injection. Serum transaminases (GOT, GPT) and serum concentrations of triiodothyronine and thyroxine were determined 28 h after the injection of galactosamine. Histology of the liver was performed by light microscopy. Our results showed that the administration of somatostatin significantly (P less than 0.02) reduced the elevation of GOT and GPT activity and diminished the degree of necrosis, and that although the administration of dibutyryl-cAMP (5 mg/100 g body wt.) intensified galactosamine induced liver injury, this effect of dibutyryl-cAMP could be completely prevented by somatostatin treatment. There was no difference in the serum concentrations of triiodothyronine and thyroxine in controls as compared to galactosamine and galactosamine plus somatostatin treated rats. At present the mechanism of this cytoprotection by somatostatin is unknown.     

Cysteine sulfinic acid decarboxylase activity in response to thyroid hormone administration in rats

The modulation of hepatic and renal cysteine sulfinic acid decarboxylase (EC 4.1.1.29) activities by triiodothyronine (T3) was studied in a series of experiments. In a dose--response study, hepatic cysteine sulfinic acid decarboxylase activity (CSAD) was depressed by 65% and renal activity was increased threefold in rats injected with 100 micrograms T3/100 g body wt for 7 days when compared to rats injected with 0.3 micrograms T3/100 g body wt. Western blot analysis indicated that these changes in CSAD activity were due to changes in the quantity of CSAD protein. Changes in hepatic and renal activities were not evident until 24 h after T3 administration. In response to T3 clearance, hepatic and renal CSAD activities approached euthyroid values 4-7 days after cessation of T3 injections although serum T3 concentrations were no different from euthyroid values 48 h after T3 injections were stopped. These data indicate that thyroid hormone effects persist after T3 clearance. The response of CSAD to thyroid status may be related to its role in taurine biosynthesis.     

Effects of L-thyroxine and L-triiodothyronine on protein and nucleic acid contents of liver of 6N-2-propylthiouracil treated hypothyroid singi fish, Heteropneustes fossilis bloch

Three consecutive injections of 12.5 X 10(-10) and 25 X 10(-10) moles/g of L-thyroxine (T4) or a single injection of L-triiodothyronine (T3) at 7.5 X 10(-10) moles/g to Singi fish caused an increase in liver protein and RNA contents, whereas similar injections of 50 X 10(-10) moles/g of T4 or 75 X 10(-10) moles/g of T3 caused a fall in these cellular constituents in liver. Treatments of Singi fish with thiourea (1 mg/ml) for 30 days caused a fall in the protein and RNA contents in liver which were restored to the euthyroid control level by a single injection of 7.5 X 10(-10) moles/g of T3 or three consecutive injections of T4 at 12.5 X 10(-10) moles/g dose. Administration of T4 (12.5 X 10(-10) moles/g, three consecutive injections) along with 6N-2-propylthiouracil (PTU) at 20 micrograms/g of b. w. in six consecutive injections to the thiourea treated (hypothyroid) fish failed to cause any change in hepatic protein and RNA contents in comparison to only PTU-treated hypothyroid fish, but a single injection of 7.5 X 10(-10) moles/g of T3 to the PTU-treated hypothyroid fish increased these cellular constituents of liver. A dose-dependent biphasic nature of thyroid hormone action, a higher potency of T3 than T4 and the probable 'prohormone' nature of T4 have been documented in case of Singi fish in the present experiments.     

Ethanol metabolism in guinea pig: In vivo ethanol elimination,alcohol dehydrogenase distribution,and subcellular localization of acetaldehyde dehydrogenase in liver

Guinea pig ethanol metabolism as well as distribution and activities of ethanol metabolizing enzymes were studied. Alcohol dehydrogenase (ADH; EC 1.1.1.1) is almost exclusively present in liver except for minor activities in the cecum. All other organ tissues tested (skeletal muscle, heart, brain, stomach, and testes) contained only negligible enzyme activities. In fed livers, ADH could only be demonstrated in the cytosolic fraction (2.94 μmol/g liver/min at 38 °C) and its apparent K_m value of 0.42 mm for ethanol as substrate is similar to the average K_m of the human enzymes. Acetaldehyde dehydrogenase (ALDH; EC 1.2.1.3) of guinea pig liver was measured at low (0.05 mm) and high (10 mm) acetaldehyde concentrations and its subcellular localization was found to be mainly mitochondrial. The total acetaldehyde activity in liver amounts to 3.56 μmol/g/ min. Fed and fasted animals showed similar zero-order alcohol elimination rates after intraperitoneal injection of 1.7 or 3.0 g ethanol/kg body wt. The ethanol elimination rate of fed animals after 1.7 g ethanol/kg body wt (2.59 μmol/g liver/min) was inhibited by 80% after intraperitoneal injection of 4-methylpyrazole. Average ethanol elimination rates in vivo after 1.7 g/kg ethanol commanded only 88% of the totally available ADH activity in fed guinea pig livers. Catalase (EC 1.11.1.6), an enzyme previously implicated in ethanol metabolism, is of 3.4-fold higher activity in guinea pig (10,400 U/g liver) than in rat livers (3,100 U/g liver), but 98% inhibition by 3-amino-1,2,4-triazole did not significantly alter ethanol elimination rates. After ethanol injection, fed and fasted guinea pigs reacted with prolonged hyperglycemia.     

The effect in vivo of alterations in gonadotropins and cyclic nucleotides on oocyte maturation in the hamster

The temporal relationship of changes in cAMP and cGMP to oocyte maturation was examined in proestrous hamsters (day 4). The first series of experiments showed, in normal cycling hamsters, an increase in cAMP and a decrease in cGMP at 1400 h shortly after the rise in LH with oocyte maturation beginning at 1800 h. When a second group of animals was injected with phenobarbital at 1200 h to block the LH surge, no significant change occurred in either cyclic nucleotide and oocyte maturation was prevented. In the second series of experiments single injections of either saline, hCG (30 IU), LH (10 micrograms) or FSH (10 micrograms) were given each to a group of animals at 0900 h on day 4. Animals were killed at five time intervals between 15 min and 3 h following the injection. LH and hCG stimulated a simultaneous increase in cAMP and decline in cGMP. The injection of FSH, however, did not cause an increase in cAMP but still produced a sharp decline in cGMP. Oocyte maturation occurred at 3 h in those animals injected with gonadotropins. Animals injected with saline showed neither cyclic nucleotide changes nor oocyte maturation. When cAMP and cGMP levels were expressed as a ratio (cAMP/cGMP) a significant increase occurred in the normal cycling animals and in those injected at 0900 h with gonadotropins. Phenobarbital and saline injected control animals showed no significant increase in the cAMP/cGMP ratio and no oocyte maturation. The results of these experiments and previous studies by this investigator indicate that cGMP may play an important role in oocyte maturation in the hamster prior to the LH surge. Since, in the presence of gonadotropins, the cAMP/cGMP ratio increases prior to oocyte maturation, it may be that the cyclic nucleotide ratio is also of importance in this process. Previous work by Hubbard and Terranova (1) has shown that guanosine 3':5' cyclic monophosphate (cGMP), can inhibit spontaneous maturation of hamster oocytes in vitro. This inhibitory action was dose dependent and overcome by LH. The cGMP-mediated inhibition occurred only in cumulus-enclosed oocytes, while adenosine 3':5' cyclic monophosphate (cAMP) inhibited spontaneous maturation in both cumulus-enclosed and denuded oocytes. The results of this study suggested that cGMP may play a role in inhibiting oocyte maturation prior to the LH surge. LH, the initiator of oocyte maturation, has also been shown in the intact proestrous rat and hamster to cause a decrease in cGMP at the same time that cAMP is rising (2,3).(ABSTRACT TRUNCATED AT 400 WORDS)     

Temporal relationship of cyclic nucleotide levels and calcium exchange to histamine-induced tension development

The purpose of these experiments was to study the temporal relationship between tension development in incubated guinea pig tracheal smooth muscle and changes in tissue levels of cAMP and cGMP, and isotopic Ca. Dose-response studies were performed with increasing concentrations of histamine both in the absence and presence of H1 receptor blockade using 10(-5) M diphenhydramine. The time course of tension development was subsequently determined in the presence of three concentrations of histamine shown to cause 50% (3 X 10(-6) M), 85% (9 X 10(-6) M), and 100% (5 X 10(-5) M) of maximal contraction. Tissue cyclic nucleotide and 45Ca levels were measured 20 sec, 1 min, and 6 min after the onset of contraction. For comparison, the influence of carbachol was also studied. Our findings demonstrate that there were no detectable alterations in tissue cAMP or cGMP levels during the initial phases of contractile change. In contrast, tissue isotopic Ca uptake increased early in histamine-induced contraction and was blocked by the H1 antagonist.     

Essential role of calcium influx in the adrenergic regulation of cAMP and cGMP in rat pinealocytes

The role of Ca2+ in the adrenergic stimulation of pinealocyte cAMP and cGMP was investigated. In this tissue alpha 1-adrenoceptor activation, which by itself is without effect, potentiates beta 1-adrenergic stimulation of cAMP and cGMP 30- to 100-fold. The present results indicate that chelation of extracellular Ca2+ with EGTA or inhibition of Ca2+ influx with inorganic Ca2+ channel blockers (La3+, Co2+, Mn2+) markedly reduces the cyclic nucleotide response to norepinephrine, a mixed alpha 1- and beta-adrenergic agonist, but not to isoproterenol, a beta-adrenergic agonist. In addition, the potentiating effects of alpha 1-adrenergic agonists were mimicked by agents which elevate cytosolic Ca2+, including K+ (EC50 = 2 X 10(-2) M), ouabain (EC50 = 2 X 10(-6) M), ionomycin (EC50 = 3 X 10(-6) M), and A23187 (EC50 = 2 X 10(-6) M); each potentiated the effects of beta-adrenergic stimulation but had no effect alone. Together these results indicate that an alpha 1-adrenoceptor-stimulated Ca2+ influx is essential for norepinephrine to increase pinealocyte cAMP and cGMP.     

Nitric oxide synthase and cGMP-mediated stimulation of renin secretion

The interaction between nitric oxide (NO) and renin is controversial. cAMP is a stimulating messenger for renin, which is degraded by phosphodiesterase (PDE)-3. PDE-3 is inhibited by cGMP, whereas PDE-5 degrades cGMP. We hypothesized that if endogenous cGMP was increased by inhibiting PDE-5, it could inhibit PDE-3, increasing endogenous cAMP, and thereby stimulate renin. We used the selective PDE-5 inhibitor zaprinast at 20 mg/kg body wt ip, which we determined would not change blood pressure (BP) or renal blood flow (RBF). In thiobutabarbital (Inactin)-anesthetized rats, renin secretion rate (RSR) was determined before and 75 min after administration of zaprinast or vehicle. Zaprinast increased cGMP excretion from 12.75 +/- 1.57 to 18.67 +/- 1.87 pmol/min (P < 0.003), whereas vehicle had no effect. Zaprinast increased RSR sixfold (from 2.95 +/- 1.74 to 17.62 +/- 5.46 ng ANG I. h(-1) x min(-1), P < 0.024), while vehicle had no effect (from 4.08 +/- 2.02 to 3.87 +/- 1.53 ng ANG I x h(-1) x min(-1)). There were no changes in BP or RBF. We then tested whether the increase in cGMP could be partially due to the activity of the neuronal isoform of NO synthase (nNOS). Pretreatment with the nNOS inhibitor 7-nitroindazole (7-NI; 50 mg/kg body wt) did not change BP or RBF but attenuated the renin-stimulating effect of zaprinast by 40% compared with vehicle. In 7-NI-treated animals, zaprinast-stimulated cGMP excretion was attenuated by 48%, from 9.17 +/- 1.85 to 13.60 +/- 2.15 pmol/min, compared with an increase from 10.94 +/- 1.90 to 26.38 +/- 3.61 pmol/min with zaprinast without 7-NI (P < 0.04). This suggests that changes in endogenous cGMP production at levels not associated with renal hemodynamic changes are involved in a renin-stimulatory pathway. One source of this cGMP may be nNOS generation of NO in the kidney.     

Interaction between L-arginine: no system and cyclooxygenase metabolic products of arachidonic acid in coronary autoregulation.

Coronary autoregulation (CA) is the intrinsic ability of the heart to maintain its nutritive blood supply constant over a wide range of perfusion pressure. This phenomenon is regulated through several control mechanisms, while metabolic and myogenic control mechanism have dominant effects. In last few years, endothelial control mechanism, which is part of metabolic control, was intensive investigated. Dominant topic of endothelial-investigation was bioregulatory L-arginine: NO system, with his effective product--nitric oxide (NO). On the other hand, cyclooxygenase metabolic pathway products of arachidonic acid plays an important role in the control of vasomotor tone of coronary arteries. For this purpose, the aim of our study was to evaluate role of L-arginine: NO system, cyclooxygenase metabolites of arachidonic acid, as well as, their interactions in the control of CA of the isolated rat heart.. In our study rat hearts autoregulate CF between 50 and 90 cm H2O of CPP. Basal release (at 60 cm H2O) of NO (as nitrite), cAMP, cGMP and HX+X (i.e. adenosine) amounted to 2.85+/-0.25 nmol/min/g wt, 29.45+/-2.22 pmol/min/g wt, 0.43+/-0.08 pmol/min/g wt and 37.50+/-2.89 nmol/min/g wt respectively. Release of NO, cAMP and cGMP were strictly parallel with CPP-CF curve, while release of adenosine (i.e. HX + X) was an inverse function of perfusion pressure. Inhibition of NOS (L-NAME, 30 micromol/l) significantly widened autoregulatory range (40-100 cm H2O), with significant reduction in CF and NO- and cGMP release, while release of cAMP was completely reversed in the presence of L-NAME. However, inhibition of cyclooxygenase didn't influence autoregulatory range, with similar changes of NO- and cAMP-release and completely inversed values of released adenosine. When L-NAME an indomethacin (an nonspecific COX-inhibitor), 3 micromol/l where added together, they exhibit interactions between these two enzymatic systems. Namely, when L-NAME was added first, indomethacin didn't influence hemodynamic effects of NOS-inhibitor. On the other hand, when COX-inhibitor was added first, L-NAME widened autoregulatory range in small manner as after control autoregulatory experiments (40-90 cm H2O). All hemodynamic changes were followed with similar changes in NO-release, what suggest that exist interaction between L-arginine: NO system and COX-metabolites in the regulation of coronary autoregulation.