Ronald J. Walkenbach

Kidney membrane cyclic AMP receptor and cyclic AMP-dependent protein kinase activities: Comparison of plasma membrane and cytoplasmic fractions

Abstract Hog kidney cortex plasma membrane and cytosol fractions were utilized for comparison of cyclic AMP receptor and cyclic AMP-dependent protein kinase activities. Both preparations exhibited relatively high affinity for cyclic 3′5′ AMP and cyclic AMP activation of the phosphorylation of protamine. In addition cyclic AMP enhanced the phosphorylation of plasma membranes. The specificity of the membrane receptor appeared to be greater than the cytosol receptor as determined by competitive binding studies. The renal plasma membrane receptor could be solubilized with the detergents Lubrol-WX or sodium deoxycholate without inhibiting receptor activity. Triton X-100 solubilized membrane protein but also reduced 3H-cyclic AMP binding activity.

Studies of kidney plasma membrane adenosine-3′,5′-monophosphate-dependent protein kinase

Abstract Porcine kidney cortex was utilized for the preparation of plasma-membrane-enriched and soluble cytoplasmic (cytosol) fractions for the purpose of examining the relative properties of cyclic [3H]AMP receptor and cyclic AMP-dependent protein kinase activities of these preparations. The affinity, specificity and reversibility of cyclic [3H]AMP interaction with renal membrane and cytosol binding sites were indicative of physiological receptors. Binding sites of cytosol and deoxycholate-solubilized membranes were half-saturated at approx. 50nM and 100 nM cyclic [3H]AMP. Native plasma membranes exhibited multiple binding sites which were not saturated up to 1 mM cyclic [3H]AMP. Modification of the cyclic phosphate configuration or 2′-hydroxyl of the ribose moiety of cyclic AMP produced a marked reduction in the effectiveness of the cyclic AMP analogue as a competitor with cyclic [3H]AMP for renal receptors. The cyclic [3H]AMP interaction with membrane and cytosol fractions was reversible and the rate and extent of dissociation of bound cyclic [3H]AMP was temperature dependent. With the plasma-membrane preparation, dissociation of cyclic [3H]AMP was enhanced by ATP or AMP. Assay of both kidney subcellular fractions for protein kinase activity revealed that cyclic AMP enhanced the phosphorylation of protamine, lysine-rich and arginine-rich histones but not casein. The potency and efficacy of activation of renal membrane and cytosol protein kinase by cyclic AMP analogues such as N6-butyryl-adenosine cyclic 3′,5′-monophosphate or N6,O2-dibutyryl-adenosine cyclic 3′,5′-monophosphate supported the observations on the effectiveness of cyclic AMP analogues as competitors with cyclic [3H]AMP in competitive binding assays. This study suggested that the membrane cyclic [3H]AMP receptors may be closely associated with the membrane-bound catalytic moiety of the cyclic AMP-dependent protein kinase system of porcine kidney.

Characterization of β-adrenergic receptors in fresh and primary cultured bovine corneal endothelium

Homogenates of fresh bovine corneal endothelium and of cells from primary cultures exhibited high affinity binding of [3H]-dihydroalprenolol, a specific beta-adrenergic antagonist. The binding was rapid and reversible. Specific binding of the radioligand in each preparation was saturable with half-maximal binding occurring at 0.5 nM. Homogenates of fresh tissue consistently showed a higher maximal binding capacity than did those from cell cultures. Both homogenates bound adrenergic agents in a manner consistent with the labelling of beta-adrenergic receptors. The relative affinities of epinephrine and norepinephrine indicated that beta-adrenergic receptors in both preparations are of the beta 2 subtype. The beta-adrenergic agonist, isoproterenol, increased the cAMP content of intact, cultured endothelial cells 27-fold over control. This effect was completely blocked by the beta-adrenergic antagonist, propranolol. Bovine corneal endothelial cells in primary culture contain beta-adrenergic receptors which are essentially identical to those from in vivo sources and appear to be linked functionally to cAMP synthesis in these cells.

Alpha1-adrenoceptors in the corneal endothelium

The potent alpha 1-adrenoceptor antagonist, [3H]prazosin, exhibited high affinity, specific and reversible binding to intact rabbit, bovine and human corneal endothelial cells in culture. The binding of 1 nM [3H]prazosin to rabbit cells reached a steady-state level within 10 min at 37 degrees C. Under these conditions, approximately 50% of the [3H]prazosin bound was specific. The level of specific [3H]prazosin binding was concentration-dependent, but Rosenthal analysis indicated that [3H]prazosin bound to at least two sites. One site exhibited a high affinity for [3H]prazosin (Kd = 0.2 nM), but a relatively low binding capacity (Bmax = 175 fmol bound mg-1 protein); the other site showed a relatively low affinity for the radioligand (Kd = 85 nM), but a much higher binding capacity (1280 fmol mg-1). Several known alpha 1-adrenoceptor antagonists and agonists competitively inhibited [3H]prazosin binding at the high affinity site when incubated with the radioligand. The relative potencies of these competing ligands were generally consistent with their binding affinities for alpha 1-adrenoceptors in other tissues. Phenylephrine stimulated the rate of hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate by 63% in these cells. This stimulation was inhibited by 52% if phentolamine was also present during the incubation. These data indicate that corneal endothelial cells have alpha 1-adrenoceptors which can modulate polyphosphoinositide turnover in this tissue.

Inhibition of adenylate cyclase activity in the corneal epithelium by anti-inflammatory steroids

Abstract Adenylate cyclase activity in bovine corneal epithelial or rat kidney particulate fractions was completely inhibited by clinically employed levels of anti-inflammatory steroids. Dose-response studies using several steroids showed that dexamethasone phosphate was the most potent enzyme inhibitor, followed respectively by prednisolone phosphate and hydrocortisone phosphate. Enzyme inhibition occurred regardless of the level or mechanism of adenylate cyclase activation, although agonist-stimulated activity was more sensitive to inhibition than fluoride-stimulated activity. Steroids appear to cause a direct, reversible and non-competitive inhibition of adenylate cyclase, affecting both the apparent substrate affinity and maximum velocity of the enzyme. Application of high levels of anti-inflammatory steroids may block the reported cyclic AMP-mediated effects of increased epithelial wound closure rates and stimulated epithelial basement membrane protein synthesis in a healing cornea.

Distribution of cyclic AMP-dependent protein kinase in the bovine cornea

Soluble and particulate fractions of bovine corneal epithelium, stroma and endothelium were prepared and assayed for protein kinase activity. All preparations exhibited measurable activity in the absence of cyclic AMP (cyclic AMP-independent activity) as well as significant enzyme stimulation in its presence (cyclic AMP-dependent activity). The epithelial layer contained over 90% of the total corneal cyclic AMP-dependent protein kinase activity, almost all of it in the soluble fraction. In contrast, over one-third of the endothelial activity was expressed by the particulate fraction. All preparations showed high affinity and high specificity for, cyclic AMP in terms of binding cyclic [3H]AMP and stimulating protein kinase activity. Elution characteristics of soluble preparations on DEAE cellulose columns demonstrated that epithelial and endothelial layers contained predominantly ‘Type II cyclic AMP-dependent protein kinase, whereas stroma had the ‘Type I enzyme. Each preparation studies also exhibited endogenous substrates for cyclic AMP-dependent protein kinase.

Characteristics of β-adrenergic receptors in bovine corneal epithelium: Comparison of fresh tissue and cultured cells

Abstract The characteristics of the β-adrenergic receptors in homogenates of fresh tissue and cultured bovine corneal epithelium were compared using [3H]dihydroalprenolol. High affinity, specific binding sites were observed in both preparations. Fresh tissue exhibited a higher binding site density (165 fmol/mg protein) than did cells in culture (57 fmol/mg protein). Studies with various β-adrenergic agonists and antagonists indicated that binding characteristics were typical of β-adrenergic receptors, predominantly of the β2 subtype. These results demonstrate that β-adrenergic receptors exist in both fresh and cultured bovine corneal epithelium and that these receptors are qualitatively and quantitatively similar.

Regulation of cyclic AMP-dependent protein kinase and glycogen synthase by cyclic AMP in the bovine cornea

Bovine corneas were treated in vitro with various drug regimens and cyclic AMP, protein kinase and glycogen synthase activities were determined in each tissue extract. Isoproterenol (alone) did not increase corneal cyclic AMP under any condition tested. The phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine (IBMX), produced a maximal 2·7-fold increase over control cyclic AMP values. Combinations of isoproterenol (or other β -adrenergic agonists) and IBMX stimulated cyclic AMP up to 25-fold over control. These increases were blocked completely by propranolol. Prostaglandin E 2 (with IBMX) was the only other drug tested which caused a significant increase (6·7-fold over control) in corneal cyclic AMP. Protein kinase activity was stimulated proportionally by cyclic AMP increases up to 8-fold over control. At this point the protein kinase activity ratio was essentially 1·0, indicating complete activation of the enzyme. Glycogen synthase activity decreased sharply in response to increases in corneal cyclic AMP and protein kinase activity. Maximal enzyme inhibition required only fourfold increases in cyclic AMP and approximately half-maximal stimulation of cyclic AMP-dependent protein kinase. These data indicate the conditions necessary to stimulate cyclic AMP in the cornea and the physiological relevance of cyclic AMP increases in terms of its effects on other corneal enzymes.

The effects of UW solution and its components on corneal thickness during and after storage

The ability of rabbit corneas to undergo energy-dependent deturgescence was examined after the corneas were stored at 4 degrees C in UW solution, M-K media, or selected modifications of these media. All corneas slowly increased in thickness during storage, despite the presence of colloidal osmotic agents. Corneas stored for 2.5 days in M-K became slightly thinner when cultured over a 24-hour period. Corneas stored in UW swelled quickly in culture and became too opaque to measure within three hours. Corneas stored in UW with 1.8 mM CaCl2 swelled transiently, then maintained their thickness and exhibited deturgescence in the latter stages of the culture period. Deturgescence of corneas stored for 7 days in M-K was only slightly worse than those stored for 2.5 days. Corneas stored for 7 days in UW, however, became opaque almost immediately in culture and those stored in calcium-supplemented UW became opaque within 4.5 hours. Replacement of the dextran in M-K with hydroxyethyl starch produced a slower rate of corneal swelling during storage and a substantially better corneal deturgescence profile during culture. Use of high concentrations of potassium ion in the M-K formulation had no significant effect on post-storage deturgescence. Replacement of glucose in M-K with the impermeable sugar, raffinose had a slight deleterious effect on corneal deturgescence in subsequent culture. Use of the impermeable anions gluconate or lactobionate to replace chloride ion caused profound corneal swelling during culture, compared with those stored in M-K. These experiments show that UW solution is inferior to M-K at preserving post-storage corneal function.(ABSTRACT TRUNCATED AT 250 WORDS)

Solubilization and photoaffinity labeling of renal membrane cyclic AMP receptors

Renal cortical plasma membranes were solubilized with sodium deoxycholate. The membrane-bound cyclic AMP receptors retained biologic activity in the detergent-dispersed state exhibiting the properties of high affinity for cyclic AMP, saturability and specificity. Half-maximal binding of cycle [3H]-AMP to these receptors was found to occur at 0.06 muM and 1.5 pmol of cyclic [3H]AMP was bound per mg membrane protein at saturation (0.5 muM cyclic [3H]AMP). Sodium deoxycholate-solubilized membrane proteins were chromatographed on Biogel A-5m. Cyclic [3H]AMP receptors eluted in the internal volume at positions equivalent to molecular sizes of 50 000 and 20 000 daltons and in the void volume at molecular size greater than 450 000. After photoaffinity labeling the renal membrane receptors with cyclic [3H]AMP, we found peaks of tritium radioactivity which eluted at similar molecular size positions on this Bogel A-5m column. Further treatment of photoaffinity labeled membranes with sodium dodecyl sulfate, mercaptoethanol and urea, followed by polyacrylamide gel electrophoresis, showed bands of tritium-labeled receptor protein with relative mobilities corresponding to molecular sizes of 26 000 and 21 000 daltons. This study shows that porcine renal cortical membranes contain at least two molecular species of cyclic AMP receptors which may be associated with regulation of the membrane-bound cyclic AMP-dependent protein kinase.