Multiple sites for interaction of prostaglandin and vasopressin in toad urinary bladder

1981 ◽  
Vol 241 (6) ◽  
pp. F625-F631
Author(s):  
D. Schlondorff ◽  
C. P. Carvounis ◽  
M. Jacoby ◽  
J. A. Satriano ◽  
S. D. Levine
Keyword(s):  
Water Flow ◽  
Protein Kinase Activity ◽  
Toad Urinary Bladder ◽  
Dependent Protein Kinase ◽  
Camp Content ◽  
Camp Generation ◽  
Dependent Protein ◽  
Endogenous Prostaglandins ◽  
Camp Levels ◽  
Inhibited Water

The interaction of vasopressin with prostaglandins were examined in the toad bladder by determining water flows, cAMP levels, and cAMP-dependent protein kinase activity. Both water flow and activation of cAMP-kinase in response to vasopressin were enhanced after prostaglandin inhibition, consistent with inhibition of vasopressin-induced cAMP generation by endogenous prostaglandins. On the other hand exogeneous PGE stimulated cAMP generation. PGE1 (10(-7) M) alone did not increase water flow but activated kinase more than vasopressin only. Addition of PGE1 (10(-7) M) and vasopressin inhibited water flow as compared with vasopressin along but increased the kinase ratio above that with vasopressin only. PGE2 (10(-5) M) increased the cAMP content and kinase ratio even more than vasopressin but again resulted in no water flow. Addition of vasopressin and PGE2 (10(-5) M) increased water flow but did not alter cAMP content or the kinase ratio compared with PGE2 alone. Similar results were obtained with PGE1. Accordingly, prostaglandin dissociates cAMP levels and kinase ratio from the hydroosmotic response, suggesting that PGE2 inhibits steps distal to cAMP. Consistent with this, in bladders pretreated with naproxen or meclofenamate, PGE2 (10(-8) to 10(-6) M) inhibited the response to submaximal doses of cAMP (5 mM) or 8-bromo-cAMP (0.03 mM). Furthermore, pretreatment with naproxen significantly enhanced the response to cAMP (5 mM). These studies provide evidence for vasopressin-PGE interaction at the site of cAMP generation and also at a step(s) unrelated to cAMP generation.

Calcium flow-independent actions of calcium channel blockers in toad urinary bladder

1983 ◽  
Vol 244 (3) ◽  
pp. C243-C249 ◽  
Author(s):  
S. D. Levine ◽  
D. N. Levin ◽  
D. Schlondorff
Keyword(s):  
Calcium Channel ◽  
Water Flow ◽  
Calcium Channel Blockers ◽  
Close Correlation ◽  
Toad Urinary Bladder ◽  
Channel Blockers ◽  
Camp Content ◽  
Calcium Blockers ◽  
Camp Generation ◽  
Transport Effects

A role for transmembrane calcium movement in vasopressin stimulation of its target cell has been postulated based on studies with calcium entry blockers such as verapamil. We examined the effect of three sets of structurally different calcium blockers--D600 (an analogue of verapamil), diltiazem, and nifedipine--on water flow in toad bladder. D600 (200 microM), diltiazem (200 microM), and nifedipine (60 microM) inhibited vasopressin-induced water flow but enhanced adenosine 3',5'-cyclic monophosphate (cAMP)-induced water flow, suggesting that the drugs inhibit cAMP generation in response to vasopressin but enhance the response to exogenous cAMP by inhibiting phosphodiesterase activity. In the case of vasopressin stimulation, inhibition of cAMP generation appears to be the overriding effect. This was confirmed by measurements of cAMP content and the protein kinase ratio (-cAMP/+cAMP), which were significantly lower in bladders receiving both D600 and vasopressin than in those receiving vasopressin alone. Furthermore the drugs inhibited activation of adenylate cyclase by vasopressin in cell homogenates and inhibited phosphodiesterase in both homogenates and membrane-free supernatants. Thus these "calcium channel blockers" can directly alter cAMP metabolism in settings where movement of calcium should be irrelevant. The close correlation between the biochemical and transport effects of these agents suggests that their effect on water flow may occur by a direct effect on cellular enzymes or the membranes in which they reside and not by altering local calcium concentrations.

Relaxing effect of pharmacologic interventions increasing cAMP in rat heart

1981 ◽  
Vol 240 (4) ◽  
pp. H441-H447
Author(s):  
L. Vittone ◽  
A. Grassi ◽  
L. Chiappe ◽  
M. Argel ◽  
H. E. Cingolani
Keyword(s):  
Protein Kinase ◽  
Rat Heart ◽  
Kinase Activity ◽  
Isolated Rat Heart ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
High Calcium ◽  
Dependent Protein ◽  
Camp Levels

The relationship between cAMP and relaxation was studied in the isolated rat heart beating at constant rate and perfused at constant coronary flow. After treatment during 1 min with different positive inotropic interventions, cyclic nucleotide levels (cAMP and cGMP) and cAMP-dependent protein kinase activity were determined in heart homogenates. Glucagon, norepinephrine, and isoproterenol increased cAMP from 0.503 +/- 0.025 pmol/mg wet wt to 1.051 +/- 0.099, 0.900 +/- 0.064, and 0.982 +/- 0.138, respectively. Simultaneously glucagon, norepinephrine, and isoproterenol increased cAMP-dependent protein kinase activity ratio from 0.21 +/- 0.02 to 0.45 +/- 0.04, 0.33 +/- 0.02, and 0.34 +/- 0.02, respectively. The ratio between maximal velocities of contraction and relaxation (+T/-T) was significantly decreased by these interventions, whereas time to peak tension (TTP) was shortened by norepinephrine and isoproterenol. High calcium, ouabain, and paired stimulation did not affect cAMP levels, TTP, or +T/-T. A striking correlation was found between cAMP-dependent protein kinase activity and relaxation induces, i.e., TTP, -T, or +T/-T (r = +/- 0.7 to -0.9). Results suggest that inotropic interventions increasing cAMP levels might be primarily affecting intracellular mechanisms causing relaxation.

Biochemical changes accompanying enhanced cardiac contractility by ionophore A23187

1985 ◽  
Vol 249 (6) ◽  
pp. H1204-H1210 ◽  
Author(s):  
J. J. Murray ◽  
P. W. Reed ◽  
J. G. Dobson
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Ionophore A23187 ◽  
Phosphorylase Activity ◽  
Dependent Protein Kinase ◽  
Camp Content ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

We have reported that the divalent cation ionophore A23187, like the beta-adrenergic agonist isoproterenol, increased the force of contraction and rate of relaxation and shortened the duration of contraction of papillary muscles isolated from guinea pigs. A23187 produced a fall in resting tension and decreased the contracture tension of K +/- depolarized muscles, as did isoproterenol. In the present studies, isoproterenol produced a concentration-dependent, rapid, and sustained increase in the cyclic AMP (cAMP) content of papillary muscle. In contrast, A23187 had no detectable effect on cAMP levels, even in the presence of the phosphodiesterase inhibitor, papaverine. Neither drug, at concentrations maximal for contractile effects, altered cyclic GMP (cGMP). Isoproterenol increased the cAMP-dependent protein kinase activity ratio, whereas A23187 did not change the activity of this enzyme. However, both A23187 and isoproterenol produced a concentration-dependent increase in phosphorylase activity. Concentrations of A23187 or isoproterenol that enhanced contractility maximally increased the alkali-labile phosphate (by ca. 35%) but were without effect on the acid-labile, alkali-stable phosphate in the total acid precipitable protein. Contractile effects of isoproterenol, which reflect activated Ca2+ uptake, and the increase in phosphorylase activity produced by this agent are believed to be due to an increase in cAMP with subsequent activation of cAMP-dependent protein kinases and phosphorylation of proteins. A23187 may produce similar contractile effects without an increase in cAMP or cAMP-dependent protein kinase activity by activating other protein kinases and/or inhibiting phosphoprotein phosphatases, most likely by its effects on intracellular calcium.

Protein synthesis inhibitors attenuate water flow in vasopressin-stimulated toad urinary bladder

1988 ◽  
Vol 254 (1) ◽  
pp. F139-F144
Author(s):  
B. S. Hoch ◽  
M. B. Ast ◽  
M. J. Fusco ◽  
M. Jacoby ◽  
S. D. Levine
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Urinary Bladder ◽  
Water Flow ◽  
Toad Urinary Bladder ◽  
Osmotic Gradient ◽  
Protein Synthesis Inhibitors ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

Vasopressin stimulates the introduction of aggregated particles, which may represent pathways for water flow, into the luminal membrane of toad urinary bladder. It is not known whether water transport pathways are degraded on removal from membrane or whether they are recycled. We examined the effect of the protein synthesis inhibitors cycloheximide and puromycin using repeated 30-min cycles of vasopressin followed by washout of vasopressin, all in the presence of an osmotic gradient, a protocol that maximizes aggregate turnover. “High dose” cycloheximide (200 micrograms/ml) inhibited flow immediately. “Low dose” cycloheximide (1 microgram/ml) did not affect initial flow; however, flow was inhibited by the fourth restimulation. On further rechallenge, inhibition persisted but did not increase. In the absence of vasopressin, inhibition did not develop. Despite the inhibition of flow in vasopressin-treated tissues, the cAMP-dependent protein kinase ratio (-cAMP/+cAMP), an index of in vivo cAMP effect, was elevated in cycloheximide-treated tissues, suggesting modulation at a distal site in the stimulatory cascade. Cycloheximide inhibited flow when 10 microM forskolin or 0.2 mM 8-BrcAMP was substituted for vasopressin in the fourth period; however, MIX (4 mM)-stimulated flow was enhanced by 1 microgram/ml cycloheximide but inhibited by 200 micrograms/ml cycloheximide. [14C]urea permeability was not inhibited by cycloheximide. Puromycin (0.5 mM) also inhibited water flow by the fourth challenge with vasopressin. The data suggest that protein synthesis inhibitors attenuate flow at a site that is distal to cAMP-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Methacholine sensitivity and cAMP protein kinase in tracheal smooth muscle

1986 ◽  
Vol 60 (3) ◽  
pp. 1043-1053 ◽  
Author(s):  
A. D. Jensen ◽  
A. M. Puckett ◽  
G. A. Rinard ◽  
T. J. Torphy ◽  
S. E. Mayer
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Cricoid Cartilage ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Camp Content ◽  
Camp Dependent Protein Kinase ◽  
Resting Tension ◽  
Dependent Protein

We studied regional variation in canine trachealis smooth muscle sensitivity and responsiveness to methacholine as well as basal and methacholine-stimulated adenosine 3′,5′-cyclic monophosphate (cAMP) and cAMP-dependent protein kinase activity. The trachea between the cricoid cartilage and the carina was divided into three segments of equal length (designated cervical, middle, and thoracic regions), each consisting of approximately 12–14 cartilage rings. Smooth muscle strips from each of the three regions were exposed to cumulative half-log increments of methacholine chloride. The sensitivity (-log EC50) and responsiveness (force per cross-sectional area and force per milligram protein) of the smooth muscle to methacholine in each region was determined from these data. Smooth muscle strips from cervical and thoracic regions were frozen before and after exposure to cumulative half-log increments of methacholine up to each region's previously determined EC50. Frozen samples were assayed for cAMP content or cAMP-dependent protein kinase activity. The relationship between resting tension and methacholine sensitivity and responsiveness were studied. For the size strips we used, 4 g resting tension set the average cervical and thoracic strips at 96 and 101% of their optimal length, respectively. The methacholine EC50 was not affected by a variation in resting tension. Sensitivity to methacholine was 7.1, 6.8, and 6.5 for cervical, middle, and thoracic regions, respectively. The responsiveness of the cervical and thoracic smooth muscle to methacholine was 16.4 and 16.3 g force/mm2, respectively, at an EC50 methacholine. Basal cAMP was lower in cervical smooth muscle than in thoracic. cAMP-dependent protein kinase activity ratios under both basal and EC50 methacholine-stimulated conditions were lower in cervical smooth muscle than in thoracic. We have observed in trachealis smooth muscle an inverse relationship between methacholine sensitivity and either cAMP or cAMP-dependent protein kinase activity. We suggest that cAMP and cAMP-dependent protein kinase play a role in the regulation of airway smooth muscle sensitivity to cholinergic agonists.

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