Enzymes of cyclic nucleotide metabolism in invertebrate and vertebrate sperm.

Sperm from several invertebrates contained guanylate cyclase activity several-hundred-fold greater than that in the most active mammalian tissues; the enzyme was totally particulate. Activity in the presence of Mn²⁺ was up to several hundred-fold greater than with Mg²⁺ and was increased 3–10-fold by Triton X-100. Sperm from several vertebrates did not contain detectable guanylate cyclase. Sperm of both invertebrates and vertebrates contained roughly equal amounts of Mn²⁺-dependent adenylate cyclase activity; in invertebrate sperm, this enzyme was generally several hundred-fold less active than guanylate cyclase. Adenylate cyclase was particulate, was unaffected by fluoride, and was generally greater than 10-fold more active with Mn²⁺ than with Mg²⁺. Invertebrate sperm contained phosphodiesterase activities against 1.0 μm cyclic GMP or cyclic AMP in amounts greater than mammalian tissues. Fish sperm, which did not contain guanylate cyclase, had high phosphodiesterase activity with cyclic AMP as substrate but hydrolyzed cyclic GMP at a barely detectable rate. In sea urchin sperm, phosphodiesterase activity against cyclic GMP was largely particulate and was strongly inhibited by 1.0% Triton X-100. In contrast, activity against cyclic AMP was largely soluble and was weakly inhibited by Triton. The cyclic GMP and cyclic AMP contents of sea urchin sperm were in the range of 0.1–1 nmol/g. Sea urchin sperm homogenates possessed protein kinase activity when histone was used as substrate; activities were more sensitive to stimulation by cyclic AMP than by cyclic GMP.⁵     

Guanylate cyclase activity and cyclic nucleotide concentrations during liver regeneration after experimental injury

Cyclic nucleotide concentrations and guanylate cyclase activity were measured in regenerating rat liver. Previous work has shown that in livers of partially hepatectomized rats the activity of a membrane-bound guanylate cyclase increases considerably during the early replicative phase [Kimura & Murad (1975) Proc. Natl. Acad. Sci. U.S.A.72, 1965-1969; Goridis & Reutter (1975) Nature (London) 257, 698-700]. Over the same time period after partial hepatectomy, increased tissue concentrations of cyclic GMP were found when the rats were killed under pentobarbital anaesthesia, but not when anaesthesia was omitted. The results obtained on hepatectomized livers were compared with the changes in guanylate cyclase activity and cyclic nucleotide concentrations during the response to galactosamine treatment. Here, a peak of guanylate cyclase activity and of cyclic GMP concentrations occurred at 8h, that is before the beginning of the proliferative response. Both parameters were normal at the time of increased DNA synthesis. There does not, therefore, seem to be a consistent correlation between changes in guanylate cyclase activity or concentrations of cyclic GMP and an increase in liver DNA synthesis. A modest rise in cyclic AMP concentrations was found, however, in livers of galactosamine-treated rats, which was coincident with the time of DNA synthesis.     

Dynamics of the change in cyclic nucleotide content in wound tissues]

Changes in the content of cyclic nucleotides (cAMP and cGMP) in the wound tissues (muscles and granulations) were studied in experiments on rats. A wound with skin defect in the dorsal area with a crushed underlying muscle served as an experimental model. It was shown that the cAMP content in the muscular tissue rises twice: during the 1st day and to a greater measure on the 7th day. The cGMP content slightly increases on the 1-4th day, drops on the 7th day and increases cAMP concentration changes similarly to that inthe muscular tissue: it increases on the 7th day and drops on the 14th day. On the contrary, the cGMP content curve in granulations is more monotonous, only a slight increase being observed on the 7th day.     

Sequential changes in the cyclic nucleotide levels and cyclic nucleotide phosphodiesterase activities during development ofMyxococcus xanthus

The levels of adenosine 3′,5′-monophosphate, guanosine 3′-5′-monophosphate and the activities of their respective phosphodiesterases exhibited changes during development ofMyxococcus xanthus that are substantially consistent with role postulated for each in a previously proposed model.     

Development of a biosensor for assaying postmortem nucleotide degradation in fish tissues

An enzyme sensor system has been developed to assess the freshness level in fish tissue. The system was designed to measure the K value, the concentration ratio of [Hx + HxR] and [Hx + HxR + IMP], where Hx, HxR, and IMP are hypoxanthine, inosine and inosine-5'-monophosphate, respectively. The [Hx + HxR] concentration in tissue extract was measured by nucleoside phosphorylase and xanthine oxidase immobilized on a preactivated nylon membrane and attached to the tip of a polarographic electrode. The electrode amperometrically detected the products of degradation, hydrogen peroxide and uric acid. For determination of [IMP + HxR + Hx], IMP was first converted to HxR by nucleotidase immobilized on the wall of a polystyrene tube. The enzyme electrode consisting of nucleoside phosphorylase and xanthine oxidase provided excellent reproducible results for at least 40 repeated assays and immobilized nucleotidase was good for at least 40 assays as well. The K value for each sample could be determined in ca. 10 min. When applied to K value measurements in several fish meats, the results obtained agreed well with those obtained by the conventional enzymatic method.     

Calmodulin-dependent cyclic nucleotide phosphodiesterase in an experimental rat model of cardiac ischemia-reperfusion

In the present study, we investigated the activity and expression of calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPDE) and the effects of calpains in rat heart after ischemia and reperfusion. Immunohistochemical studies indicated that CaMPDE in normal heart is localized in myocardial cells. Rat ischemic heart showed a decrease in CaMPDE activity in the presence of Ca2+ and calmodulin; however, in ischemic-reperfusion tissue a progressive increase in Ca2+ and calmodulin-independent cyclic nucleotide phosphodiesterase (CaM-independent PDE) activity was observed. Perfusion of hearts with cell-permeable calpain inhibitor suppressed the increase of Ca2+ and CaM-independent PDE activity. Protein expression of CaMPDE was uneffected by hypoxic injury to rat myocardium. The purified heart CaMPDE was proteolyzed by calpains into a 45 kDa immunoreactive fragment in vitro. Based on these results, we propose that hypoxic injury to rat myocardium results in the generation of CaM-independent PDE by calpain mediated proteolysis, allowing the maintenance of cAMP concentrations within the physiological range.     

Free amino acids in rat ocular tissues during postnatal development

Amino acid changes in the retina, vitreous, lens, iris-ciliary body and cornea of the rat eye were determined during postnatal growth. The amino acid concentrations of the ocular tissues showed varying profiles at various developmental stages. These results suggest a different timetable for development of each ocular tissue or indicate a synthesis of specific proteins in the postnatal period. Adult amino acid levels appeared to be fully reached on the 30th day after birth at the latest. Quantitatively the greatest changes were observed in taurine concentrations, which increased in all five ocular tissues during maturation. GABA changes paralleled those of taurine in the retina, whereas in the other ocular tissues GABA changes were very low. The greatest decrease in glutamic acid and aspartic acid concentration during postnatal development was in the lens, where these amino acids probably are needed for the synthesis of the lenticular proteins, the alpha-, beta-, and gamma-crystallines.     

Changes in cyclic nucleotide levels during embryonic development of chick hearts

The effects of isoproterenol, acetylcholine (Ach), and adenosine, on cyclic AMP (cAMP) and cyclic GMP (cGMP) contents were examined in chick hearts at various stages of embryonic development. The basal cAMP content was highest (87.7 +/- 1.3 pmol/mg protein) in young (3-day) embryonic chick hearts and decreased during development (9.6 +/- 0.6 pmol/mg protein in 9-19-day-old hearts). On the other hand, the cGMP content was lowest (45.5 +/- 2.3 fmol/mg protein) in young (3-day) embryonic chick hearts and increased during development (338 +/- 15.0 fmol/mg protein in 14-19-day-old hearts). Iso increased the cAMP concentration in embryonic hearts at all ages. Ach and Ado had no effect on the cAMP content at all ages. However, the Isoproterenol-induced stimulation of cAMP was inhibited by Ach and Adenosine at all ages. In young embryonic hearts, Ach and Ado increased cGMP concentration only slightly, whereas these agents caused a substantial increase in cGMP concentration in the older hearts. Thus, there was a clear age difference in the effects of Ach and Adenosine on the cGMP and cAMP concentrations. Nitroprusside and hydrogen peroxide increased cGMP concentration in older hearts (greater than 5-day-old) but not in the 3-day-old embryonic hearts. Thus, guanylate cyclase activity may be low in young (3-day-old) hearts. It summary, the cGMP level is very low in young embryonic chick hearts, and increases markedly during development. The changes in cGMP are reciprocal to those of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Propranolol enhances adenine nucleotide degradation in human muscle during exercise

Eight healthy men cycled to exhaustion [4.1 +/- 0.3 (SE) min] during beta-adrenoceptor blockade (beta B) with propranolol. The exercise was repeated on another day with the same power output and duration but without propranolol (control). The total adenine nucleotide (TAN) content in muscle (quadriceps femoris) decreased during exercise, and the decrease was more pronounced during beta B (delta TAN = 4.8 +/- 1.0 mmol/kg dry wt) than during control (delta TAN = 2.8 +/- 0.9; P less than 0.01, beta B vs. control). The decrease in TAN corresponded with a similar increase in inosine 5'-monophosphate (IMP). The increase in IMP was more pronounced during beta B (delta IMP = 5.1 +/- 1.2 mmol/kg dry wt) than during control (delta IMP = 2.8 +/- 0.7; P less than 0.05, beta B vs. control). Similarly, the increase in the content of NH3 in muscle was twice as high during beta B vs. control (P less than 0.01). The increase in muscle lactate and the decrease in phosphocreatine during exercise were similar between treatments, but postexercise hexose phosphates were approximately twofold higher (P less than 0.05) during control than during beta B. It is concluded that beta B enhances the degradation of TAN and the production of NH3 and IMP in muscle during intense exercise. This indicates that the imbalance between the rates of utilization and resynthesis of ATP is more pronounced during beta B possibly because of a decreased O2 transport to the contracting muscle and a diminished activation of glycolysis by the hexose phosphates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenine nucleotide degradation in human skeletal muscle during prolonged exercise

Eight healthy men cycled at a work load corresponding to approximately 70% of maximal O2 uptake (VO2max) to fatigue (exercise I). Exercise to fatigue at the same work load was repeated after 75 min of rest (exercise II). Exercise duration averaged 65 and 21 min for exercise I and II, respectively. Muscle (quadriceps femoris) content of glycogen decreased from 492 +/- 27 to 92 +/- 20 (SE) mmol/kg dry wt and from 148 +/- 17 to 56 +/- 17 (SE) mmol/kg dry wt during exercise I and II, respectively. Muscle and blood lactate were only moderately increased during exercise. The total adenine nucleotide pool (TAN = ATP + ADP + AMP) decreased and inosine 5'-monophosphate (IMP) increased in the working muscle during both exercise I (P less than 0.001) and II (P less than 0.01). Muscle content of ammonia (NH3) increased four- and eight-fold during exercise I and II, respectively. The working legs released NH3, and plasma NH3 increased progressively during exercise. The release of NH3 at the end of exercise II was fivefold higher than that at the same time point in exercise I (P less than 0.001, exercise I vs. II). It is concluded that submaximal exercise to fatigue results in a breakdown of the TAN in the working muscle through deamination of AMP to IMP and NH3. The relatively low lactate levels demonstrate that acidosis is not a necessary prerequisite for activation of AMP deaminase. It is suggested that the higher average rate of AMP deamination during exercise II vs. exercise I is due to a relative impairment of ATP resynthesis caused by the low muscle glycogen level.     

Neurotrophic control of cyclic nucleotide levels during muscle differentiation in cell culture

The effects of chick brain–spinal cord extract on morphological development and cyclic nucleotide levels of cultured chick embryo skeletal muscle cells were determined. It had previously been shown that the extract stimulated morphological differentation, protein synthesis, and cholinesterase activity of muscle cells. Myoblasts fused earlier and an increase in number as well as diameter of myotubes were seen in the extract treated cultures. Cyclic nucleotides levels were higher (almost twice the controls for both adenosine 3′, 5′ -cyclic monophosphate and guanosine 3′, 5′ -cyclic monophosphate) and preceded their occurence in the control cultures. It was suggested that factor(s) in the extract interact with membrane receptor(s) to alter nucleotide levels which, in turn, allow the effects to be expressed.     

Changes in cyclic nucleotide levels during erythroid differentiation in Friend leukemia cells

Changes in cyclic nucleotide levels were observed in Friend leukemia cells exposed to the inducers of erythroid differentiation dimethylsulfoxide (DMSO), sodium butyrate and hypoxanthine. The most extensive changes in cAMP levels occurred during the initial 24 h growth period. In uninduced cultures, cAMP concentration increased 35-fold compared with a 7-fold increase in the presence of inducer. During the same period. cGMP levels rose about 3-fold in uninduced cells compared with a 20-fold increase in induced cells.     

Zinc deficiency decreases the activity of calmodulin regulated cyclic nucleotide phosphodiesterases in vivo in selected rat tissues

The effect of zinc deficiency on calmodulin function was investigated by assessing the in vivo activity of two calmodulin regulated enzymes, adenosine 3′,5′-monophosphate (c-AMP) and guanosine 3′,5′-monophosphate (c-GMP) phosphodiesterase (PDE) in several rat tissues. Enzymatic activities in brain, heart, and testis of rats fed a zinc deficient diet were compared with activities in these tissues from pair fed, zinc supplemented rats. In testis, a tissue in which zinc concentration decreased with zinc deficient diet, enzyme activities were significantly decreased over those in rats who were pair fed zinc supplemented diets. In brain and heart, tissues in which zinc concentrations did not change with either diet, enzymatic activities between the groups were not different. These results indicate that zinc deficiency influences the activity of calmodulin-regulated phosphodiesterases in vivo supporting the hypothesis that zinc plays a role in calmodulin function in vivo in zinc sensitive tissues.     

Neurotrophic control of cyclic nucleotide levels during muscle differentiation in cell culture.

The effects of chick brain-spinal cord extract on morphological development and cyclic nucleotide levels of cultured chick embryo skeletal muscle cells were determined. It had previously been shown that the extract stimulated morphological differentiation, protein synthesis, and choliniesterase activity of muscle cells. Myoblasts fused earlier and an increase in number as well as diameter of myotubes were seen in the extract treated cultures. Cyclic nucleotides levels were higher (almost twice the controls for both adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate) and preceded their occurrence in the control cultures. It was suggested that factor(s) in the extract interact with membrane receptor(s) to alter nucleotide levels which, in turn, allow the effects to be expressed.     

Predominance of Th1 cells in ocular tissues during herpetic stromal keratitis.

Herpetic stromal keratitis (HSK) appears to represent an immunopathologic response in the cornea of the eye to HSV-1. T cells of the CD4+ subset were shown to be involved in the mediation of HSK, but how they subserve an immunopathologic role is uncertain. In the present report, we have isolated cells from eyes in the active phase of HSK and studied their cytokine profile after culture in vitro or stimulation with Ag or nonspecific mitogens. Inflammatory cells recovered from eyes consist of polymorphonuclear leukocytes, macrophages, and lymphocytes. As reported before, all the lymphocyte recovered were of the CD4+ phenotype. After stimulation in vitro with Ag or mitogen the cytokines IL-2, IFN-gamma, and TNF-alpha/beta were produced, but not the cytokines IL-4 and IL-10. Thus, on the basis of cytokine profile, ocular lymphocytes were identified as Th1 cells. Ocular cells were also stimulated with PMA and shown to produce IL-1. The results were discussed in terms of the possible means by which the Th1 cells induce tissue damage in HSK as well as in terms of the possible means by which a preferential accumulation of Th1 cell occurs in the eye.     

Functional compartments in cyclic nucleotide action

Cyclic AMP-dependent protein kinase (cAMP-PK) is a ubiquitous enzyme that, when activated by cAMP, is capable of phosphorylating a variety of intracellular proteins. The central postulate of cAMP-mediated hormone action is that hormones regulate intracellular cAMP concentration and cAMP-PK mediates the effects of this second messenger. Although this postulate accurately describes cAMP action in certain systems, it does not adequately provide for recent observations of the accumulation of cAMP and the activation of protein kinase without the anticipated effects on protein kinase's substrates. Both biochemical and cytochemical technics provide evidence that hormonally-specific regulation of cAMP action occurs and is important. Our thesis is that hormonal regulation of metabolic events via cAMP is localized intracellular phenomenon. We propose that occupation of some cell-surface hormone receptors leads to cAMP accumulation and the activation of protein kinase in subcellular compartments, with the consequent phosphorylation of specific, rather than all, substrates of protein kinase. circumstances potentially contributing to this specificity include: (a) physical and kinetic compartmentation of hormone-receptor-adenylate cyclase complexes non-randomly within the cell membrane; and, (b) a fixed spatial relationship of hormonally activated adenylate cyclase and specific intracellular regions by the participation of cytoskeletal proteins.     

Activity of enzymes involved in pyrimidine nucleotide synthesis during uterine growth in cyclic rats

The pyrimidine nucleotide salvage enzymes, uridine and thymidine kinases, and the enzyme, aspartate carbamyltranferase, involved in de novo synthesis, were investigated to assess their relative contributions to the RNA and DNA precursor requirements of uterine growth in cyclic rats. Only aspartate carbamyltransferase reflected the known fluctuations in plasma oestradiol-17beta and uterine mitotic activity, being minimal at dioestrus 1 and maximal at pro-oestrus. Treatment of ovariectomized rats with oestradiol-17beta stimulated carbamyltransferase activity, but cycloheximide prevented this response, indicating the association between the enzyme and this hormone. Uridine kinase activity did not vary during the oestrous cycle, but there were peaks of thymidine kinase activity on the days of pro-oestrus and the 1st day of dioestrus.