Interactions between kinases and phosphatases in the rapid control of brain aromatase

Aromatization of testosterone into oestradiol plays a key role in the activation of male sexual behaviour in many vertebrate species. Rapid changes in brain aromatase activity have recently been identified and the resulting changes in local oestrogen bioavailability could modulate fast behavioural responses to oestrogens. In quail hypothalamic homogenates, aromatase activity is down-regulated within minutes by calcium-dependent phosphorylations in the presence of ATP, MgCl2 and CaCl2 (ATP/Mg/Ca). Three kinases (protein kinases A and C and calmodulin kinase; PKA, PKC and CAMK) are potentially implicated in this process. If kinases decrease aromatase activity in a reversible manner, then it would be expected that the enzymatic activity would increase and/or return to baseline levels in the presence of phosphatases. We showed previously that 0.1 mM vanadate (a general inhibitor of protein phosphatases) significantly decreases aromatase activity but specific protein phosphatases that could up-regulate aromatase activity have not been identified to date. The reversibility of aromatase activity inhibition by phosphorylations was investigated in the present study using alkaline and acid phosphatase (Alk and Ac PPase). Unexpectedly, Alk PPase inhibited aromatase activity in a dose-dependent manner in the presence, as well as in the absence, of ATP/Mg/Ca. By contrast, Ac PPase completely blocked the inhibitory effects of ATP/Mg/Ca on aromatase activity, even if it moderately inhibited aromatase activity in the absence of ATP/Mg/Ca. However, the addition of Ac PPase was unable to restore aromatase activity after it had been inhibited by exposure to ATP/Mg/Ca. Taken together, these data suggest that, amongst the 15 potential consensus phosphorylation sites identified on the quail aromatase sequence, some must be constitutively phosphorylated for the enzyme to be active whereas phosphorylation of the others is involved in the rapid inhibition of aromatase activity by the competitive effects of protein kinases and phosphatases. Two out of these 15 putative phosphorylation sites occur in an environment corresponding to the consensus sites for PKC, PKA (and possibly a CAMK) and, in all probability, represent the sites whose phosphorylation rapidly blocks enzyme activity.     

Ca2+ and calmodulin-regulated endogenous tubulin kinase activity in presynaptic nerve terminal preparations

Synaptosomal tubulin was shown to be the major substrate for a Ca2+-calmodulin regulated protein kinase in synaptosome soluble fractions as determined by two-dimensional gel electrophoresis and peptide mapping. Ca2+ activated this endogenous tubulin kinase system in presynaptic nerve terminal preparations. The Ca2+-dependent activation of the tubulin kinase system was mediated by the Ca2+ binding protein, calmodulin. Trifluoperazine, a known inhibitor of calmodulin, significantly blocked the calmodulin-stimulated [32P]phosphate incorporation into synaptic tubulin. This inhibition of endogenous tubulin phosphorylation could be reversed by addition of exogenous calmodulin to the reaction mixture. The concentrations of Ca2+ and calmodulin required to produce a half-maximal stimulation of the tubulin kinase were 0.8 microM and 0.3 microM respectively. Greater than 70% of soluble tubulin present in the nerve terminal was phosphorylated in less than 50 s by this kinase system. Evidence is presented indicating that the synaptic Ca2+-calmodulin tubulin kinase is a distinct enzyme system from the previously described cyclic AMP microtubule-associated kinase. The anticonvulsant phenytoin inhibited the Ca2+-calmodulin stimulated phosphorylation of tubulin, and alpha- and beta-tubulin were identified as major components of previously designated synaptic phosphoprotein bands of DPH-L and DPH-M. Existence of the kinase as a calmodulin-tubulin-kinase complex is suggested from kinetic studies. The Ca2+-calmodulin tubulin kinase is very labile and specialized isolation procedures were necessary to retain activity. The activation of the tubulin kinase by Ca2+ and calmodulin may play a role in the functional utilization of tubulin in the nerve terminal and may mediate some of the effects of Ca2+ on synaptic function.     

Ca and calmodulin-regulated endogenous tubulin kinase activity in presynaptic nerve terminal preparations

Synaptosomal tubulin was shown to be the major substrate for a Ca²⁺-calmodulin regulated protein kinase in synaptosome soluble fractions as determined by two-dimensional gel electrophoresis and peptide mapping. Ca²⁺ activated this endogenous tubulin kinase system in presynaptic nerve terminal preparations. The Ca²⁺-dependent activation of the tubulin kinase system was mediated by the Ca²⁺ binding protein, calmodulin. Trifluoperazine, a known inhibitor of calmodulin, significantly blocked the calmodulin-stimulated [³²P]phosphate incorporation into synaptic tubulin. This inhibition of endogenous tubulin phosphorylation could be reversed by addition of exogenous calmodulin to the reaction mixture. The concentrations of Ca²⁺ and calmodulin required to produce a half-maximal stimulation of the tubulin kinase were 0.8 μM and 0.3 μM respectively. Greater than 70% of soluble tubulin present in the nerve terminal was phosphorylated in less than 50 s by this kinase system. Evidence is presented indicating that the synaptic Ca²⁺-calmodulin tubulin kinase is a distinct enzyme system from the previously described cyclic AMP microtubule-associated kinase. The anticonvulsant phenytoin inhibited the Ca²⁺-calmodulin stimulated phosphorylation of tubulin, and α- and β-tubulin were identified as major components of previously designated synaptic phosphoprotein bands DPH-L and DPH-M. Existence of the kinase as a calmodulin-tubulin-kinase complex is suggested from kinetic studies. The Ca²⁺-calmodulin tubulin kinase is very labile and specialized isolation procedures were necessary to retain activity. The activation of the tubulin kinase by Ca²⁺ and calmodulin may play a role in the functional utilization of tubulin in the nerve terminal and may mediate some of the effects of Ca² on synaptic function.     

Ca2+ activated and pH sensitive cyclic AMP phosphodiesterase in the nervous system of the mollusc Pleurobranchaea

Regulation of cyclic AMP through its synthesis is known to be important in modulating the activity of molluscan neurons; however, no data exists regarding the regulation of cyclic AMP degradation. We find that cyclic AMP phosphodiesterase (PDE) activity in homogenates of the nervous system of the mollusc Pleurobranchaea is significantly stimulated by calcium ion. Ca2+ stimulation is suppressed by the calmodulin antagonist trifluoperazine (TFP), indicating resemblance to the Ca2+-calmodulin PDEs of mammalian neurons. Ca2+ also accentuates the pH sensitivity of PDE. The qualities of Ca2+ and pH sensitivity of PDE are fitted into a model for cAMP regulation of neuronal activity in an identified feeding command neuron; the postulated role of PDE is consistent with effects of cAMP, TFP, and pH on the neuron's activity.     

Distribution and regulation of estrogen-2-hydroxylase in the quail brain

The anatomical distribution and endocrine regulation of the estrogen-2-hydroxylase activity were investigated in the brain of adult male and female Japanese quail. Significant levels of enzymatic activity were detected in all brain regions that were studied, but the highest levels were observed in preoptic and hypothalamic brain nuclei that are known to contain high levels of aromatase activity. These data are consistent with previous results suggesting that the placental aromatase is also responsible for the estrogen-2-hydroxylase activity. However, there is a marked sex difference and a control by T of aromatase activity in the quail brain, and no such difference in 2-hydroxylase activity could generally be detected except in the VMN. Further studies will be needed to know whether the previously published conclusions concerning the human placenta also apply to the brain. The present data are consistent with the idea that estrogens formed locally in the brain by testosterone aromatization could affect reproduction by interfering with the catecholaminergic transmission after being metabolized into catechol-estrogens.     

Neuroanatomical specificity in the autoregulation of aromatase-immunoreactive neurons by androgens and estrogens: an immunocytochemical study

Testosterone (T) increases brain aromatase activity (AA) in quail and other avian and mammalian species. It was shown both in quail and in rat that this enzymatic induction results from a synergistic action of androgens and estrogens. These studies provide little information on possible anatomical or cellular specificity of the effect. Using a polyclonal antiserum against human placental aromatase, we have previously identified aromatase-immunoreactive (ARO-ir) neurons in the quail brain and demonstrated that T increases the number of ARO-ir cells in the quail preoptic area (POA) supporting previous evidence that T increases AA in the brain. However, which T metabolites are involved, the actual mechanism of regulation and the possibility of anatomical specificity for these effects are not yet clear. In the present study, we disassociated the effects of androgens and estrogens in aromatase induction by comparing ARO-ir neurons of quail treated with T alone or T in the presence of a potent aromatase inhibitor (R76713), which has been shown to depress AA levels and to suppress T-activated copulatory behavior. T increased the number of ARO-ir cells in POA, bed nucleus striae terminalis (BNST) and tuberal hypothalamus (Tu). The T effect was inhibited by concurrent treatment with aromatase inhibitor in Tu, but not in POA and BNST. This differential effect of the aromatase inhibitor fits in very well with our previous studies of the co-localization of aromatase and estrogen receptors. The T effect was blocked by R76713 in areas where ARO-ir and estrogen receptor-ir are generally co-localized (Tu) and was not affected in areas with mainly ARO-ir positive, estrogen receptor-ir negative cells (POA, BNST). This suggests anatomical differences in the expression or clearance of aromatase which may be differentially sensitive to androgens and estrogens and dependent upon the presence of sex steroid receptors.     

Immunohistochemical determination of calcium-calmodulin binding predicts neuronal damage after global ischemia

Since ionic Ca2+ binds with intracellular calmodulin (CaM) before activating proteases, kinases, and phospholipases, demonstration of persistent Ca2+-CaM binding in neurons destined to show ischemic cellular injury would support the concept that elevated intracellular Ca2+ plays a causative role in ischemic neuronal damage. In order to characterize Ca2+-CaM binding, we used a sheep anti-CaM antibody (CaM-Ab) which recognizes CaM that is not bound to Ca2+ or brain target proteins. Therefore, immunohistochemical staining of brain sections by labeled CaM-Ab represented only unbound CaM. Six normal rats were compared to 15 animals rendered ischemic for 30 min by a modification of the four-vessel occlusion model. Animals were killed immediately after ischemia, and after 2 and 24 h of reperfusion. Brain sections through hippocampus were incubated in CaM-Ab, and a diaminobenzadiene labeled anti-sheep secondary antibody was added to stain the CaM-Ab. Staining in the endal limb of dentate, dorsal CA1, lateral CA3, and parietal cortex was graded on a 4-point scale. All normal animals had grade 4 staining indicating the presence of unbound CaM in all four brain regions. Ischemic animals demonstrated reduced (grade 0 to 2) staining in the CA1 and CA3 regions immediately and 2 and 24 h after ischemia (p less than 0.01 for both regions at all three time intervals) indicating persistent binding of CaM with Ca2+ and target proteins in these regions. Staining decreased in dentate and cortex up to 2 h after ischemia (p = 0.02 for both regions) but returned toward normal by 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical localization of ionotropic glutamate receptors subunits in the adult quail forebrain

The excitatory amino acid glutamate is implicated in the central control of many neuroendocrine and behavioral processes. The ionotropic glutamate receptors are usually divided into the N-methyl-D-aspartate (NMDA) and non-NMDA (kainate and AMPA) subtypes. Subunits of these receptors have been cloned in a few mammalian species. Information available in birds is more limited. In quail, we recently demonstrated that glutamate agonists (kainate, AMPA, and NMDA) rapidly (within minutes) and reversibly decrease in vitro aromatase activity like several other manipulations affecting intracellular HCa(2+) pools. Aromatase catalyzes the conversion of androgens into estrogens which is a limiting step in the control by testosterone of many behavioral and physiologic processes. Therefore, glutamate could control estrogen production in the brain, but the anatomic substrate supporting this effect is poorly understood. In quail, aromatase is mainly localized in the preoptic-hypothalamic-limbic system. We visualized here the distribution of the major ionotropic glutamate receptors in quail by immunocytochemical methods by using commercial primary antibodies raised against rat glutamate receptor 1 and receptors 2-3 (GluR1, GluR2/3: AMPA subtype, Chemicon, CA), rat glutamate receptors 5-7 (GluR5-7: kainate subtype, Pharmingen, CA), and rat NMDA receptors (NMDAR1, Pharmingen, CA). Dense and specific signals were obtained with all antibodies. The four types of receptors are broadly distributed in the brain, and, in particular, immunoreactive cells are identified within the major aromatase cell groups located in the medial preoptic nucleus, ventromedial hypothalamus, nucleus striae terminalis, and nucleus taeniae. Dense specific populations of glutamate receptor-immunoreactive cells are also present with a receptor subtype-specific distribution in broad areas of the telencephalon. The distribution of glutamate receptors, therefore, is consistent with the idea that these receptors could be located at the surface of aromatase-containing cells and mediate the rapid regulation of aromatase activity in a direct manner.     

Calcium-dependent interactions of the human norepinephrine transporter with syntaxin 1A

The norepinephrine (NE) transporter (NET) terminates noradrenergic signaling by clearing released NE at synapses. The activity of NET can be rapidly regulated by depolarization and receptor activation via Ca2+ and kinase/phosphatase-linked pathways. The SNARE protein syntaxin 1A (SYN1A) interacts with NET and influences transporter surface trafficking and catalytic activity. In this study, we establish a link between changes in intracellular Ca2+ and SYN1A/NET interactions. SYN1A influenced NE transport only in the presence of Ca2+ in brain cortical synaptosomes. Although NET/SYN1A associations were sensitive to manipulations of Ca2+ in CHO cells, in vitro binding experiments using purified NET and SYN1A fusion proteins demonstrated a lack of direct Ca2+ sensitivity. Disruption of NET/SYN1A interaction abolished inhibition of NE transport by phorbol ester (PMA) to activate protein kinase C (PKC), but had no effect on transport inhibition by the Ca2+ calmodulin kinase (CaMK) inhibitor KN93. Furthermore, PMA enhanced Ca2+-dependent modulation of NE transport in synaptosomes. Our data reveal roles for SYN1A in the Ca2+-dependent regulation of NET, likely reliant on regulation by PKC signaling, but independent of CaMK.     

In situ protein phosphorylation in hippocampal tissue slices

We have studied the subcellular distribution of phosphoproteins in intact hippocampal slices and examined factors that regulate their phosphorylation and dephosphorylation in situ. The presence of Ca2+ in slice equilibration and prelabeling buffers and high-K+-induced depolarization markedly increased 32Pi incorporation into endogenous proteins. Ca2+-stimulatory effects were significantly reduced by Ca2+-channel blockers and the calmodulin antagonist W-13. Certain proteins were dephosphorylated in situ, and their dephosphorylation was dependent on both Ca2+ and depolarization. A number of proteins phosphorylated in situ was similar to those previously characterized in synaptic fractions phosphorylated in vitro. Many phosphoproteins were identified on the basis of molecular weight, isoelectric point, immunoreactivity, and phosphopeptide mapping; these included the 87 kDa substrate of protein kinase C, synapsin I, the 50 and 60 kDa subunits of Ca2+/calmodulin-dependent protein kinase II (CKII), tubulin, B-50, the alpha-subunit of pyruvate dehydrogenase and myelin basic proteins. CKII phosphorylation in situ appeared similar but not identical to its in vitro counterpart. Phosphopeptide mapping analysis of in situ labeled substrate proteins indicated that cAMP-, Ca2+/calmodulin-, and Ca2+/phospholipid-dependent protein kinases were all active in slice preparations under basal conditions. Increased 32Pi labeling of hippocampal proteins following tissue depolarization appeared to be associated with increased activity of endogenous protein kinases since depolarization did not result in 32Pi-labeling of any new phosphoproteins.     

Calcium-binding proteins in whole brain and synaptic subfractions

At least 19 calcium-binding proteins were detected in avian brain subfraction using 45Ca2+ binding to proteins immobilized in polyacrylamide gels. Half of the 45Ca2+ binding proteins were observed in presynaptic cytoplasm. Two-dimensional gel electrophoresis of this material revealed at least 14 45Ca2+ binding polypeptides besides calmodulin. These proteins may be important in brain and nerve terminal function.     

Inorganic lead and calcium interact positively in activation of calmodulin

Calmodulin is a ubiquitous calcium-binding protein that mediates many of the intracellular actions of Ca2+ ions. The calcium-binding sites of calmodulin consist of four EF-hand motifs; full activation of calmodulin normally occurs when all four sites are occupied by Ca2+. Inorganic lead (PY2+) has been shown to activate calmodulin at total lead concentrations similar to the concentrations of Ca2+ required for activation (Goldstein and Ar, 1983; Habermann et al., 1983), but the free Pb2+ concentrations required for calmodulin activation have not been determined. In addition, it is possible that activation may occur with different sites occupied by different divalent cations, for example Ca2+ and Pb2+. We investigated the ability of free Pb2+, alone or in combination with Ca2+, to activate calmodulin. In aqueous media, N-phenyl-1-naphthylamine (NPN) and 8-anilino-1-naphthalenesulfonate (ANS) show increased fluorescence when bound to hydrophobic regions of proteins. This increased fluorescence has been used to monitor the conformational change that occurs during calmodulin activation (LaPorte et al., 1980). In the presence of calmodulin, both Ca2+ and Pb2+ stimulated increased fluorescence of NPN and ANS. Threshold and EC50 free metal concentrations were approximately 100 nM and 450-500 nM, respectively, for Ca2+ and 100 pM and 400-550 pM, respectively, for Pb2+. Fluorescence was enhanced by combinations of low concentrations of free Ca2+ and Pb2+; for example, as little as 20 pM free Pb2+ enhanced fluorescence in combination with 200 nM free Ca2+. The activity of the PDE1 isoform of cyclic nucleotide phosphodiesterase is stimulated by Ca2+/calmodulin (Wang et al., 1990). In the presence of calmodulin, we found that Ca2+ and Pb2+ activated calmodulin-stimulated PDE activity, with threshold and EC50 free metal concentrations of approximately 200 nM and 1200 nM, respectively, for Ca2+ and 300 pM and 430 pM, respectively, for Pb2+. PDE activity was stimulated by combinations of Ca2+ and Pb2+. For example, with 100 nM free Ca2+, as little as 50 to 100 pM free Pb2+ further stimulated PDE activity; with 1000 nM free Ca2+, 20 to 50 pM free Pb2+ further stimulated PDE activity. Isobolographic analysis indicated that stimulation of PDE by Ca2+ and Pb2+ was additive. These results show that concentrations of free Pb2+ as low as 100 to 300 pM activate calmodulin and that, in the presence of physiological concentrations of free Ca2+, Pb2+ can activate calmodulin at concentrations below 50 pM. The intracellular free Ca2+ concentration in Ca2+ "hot spots," for example near sites of influx through Ca2+-permeable plasma membrane channels, can reach dozens of pM, with the free Ca2+ concentration decreasing rapidly with distance from the source of the hot spot. Our results suggest that picomolar concentrations of intracellular free Pb2+ should expand both the effective amplitude and volume of Ca2+ hot spots with respect to calmodulin activation, and thus may amplify intracellular Ca2+ signaling in lead-exposed cells.     

Characteristics of a Ca2+/calmodulin-dependent binding of the Ca2+ channel antagonist, nitrendipine, to a postsynaptic density fraction isolated from canine cerebral cortex

Synaptic membrane (SM) and postsynaptic density (PSD) fractions isolated from the cerebral cortex (CTX) and cerebellum (CL) of the canine brain were found to contain one class of specific nitrendipine binding sites. The specific binding constants were: CTX-SM, Kd = 110 pM (Bmax = 126 fmol/mg protein); CTX-PSD, Kd = 207 pM (Bmax = 196 fmol/mg); CL-SM, Kd = 100 pM (Bmax = 65 fmol/mg); CL-PSD, Kd = 189 pM (Bmax = 80 fmol/mg). Treatment of the CTX-SM and CTX-PSD fractions with 0.5% deoxycholate and 1.0% N-lauroyl sarcosinate removed 88-91% and 42-51% of the nitrendipine binding, respectively, indicating that the major nitrendipine binding present in the SM fractions are of non-synaptic origin. Moreover, the percentages of total protein and specific nitrendipine binding removed from PSDs by these detergents were similar, indicating no preferential dissociation of the latter, and suggesting that the receptor protein is firmly bound and is probably an intrinsic component of the PSD fraction. Both Ca2+ and calmodulin were found to be important for the binding of nitrendipine to the CTX-SM and CTX-PSD fractions since: R24571, a calmodulin antagonist, was found to inhibit nitrendipine binding to the CTX-SM and CTX-PSD fractions with IC50 values of 1.1 microM and 0.9 microM, respectively; removal of Ca2+ from the CTX-SM and CTX-PSD fractions with 0.2 mM EGTA resulted in losses of specific nitrendipine binding of 80 and 90%, respectively; Ca2+ alone restored nitrendipine binding to EGTA-pretreated CTX-SM fractions and not to CTX-PSD fractions, with the latter needing both Ca2+ and calmodulin to restore nitrendipine binding; EGTA treatment removed 14-16% and 89-91% of nitrendipine bound to the CTX-SM and CTX-PSD fractions, respectively, suggesting that calmodulin (but not Ca2+) is needed to maintain the nitrendipine-nitrendipine receptor-calmodulin complex; Ca2+-reconstituted EGTA-pretreated CTX-SM fractions and the Ca2+ plus calmodulin-reconstituted EGTA-pretreated CTX-SM and CTX-PSD fractions were found to have similar binding constants to those for the corresponding native, untreated fractions; and the Ca2+/calmodulin dependency on nitrendipine binding was similar to the well-known Ca2+/calmodulin dependency on phosphorylation in EGTA-pretreated PSD fractions. It needed much less Ca2+ to saturate Ca2+/calmodulin-dependent phosphorylation of the pretreated CTX-PSD fractions than the nitrendipine binding. Yet, less calmodulin was needed to saturate nitrendipine binding than the phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calmodulin-dependent adenylate cyclase in rat retina and the response to dopamine

Adenylate cyclase activity from the rat neural retina was highly stimulated with Ca²⁺ and calmodulin. The retinal adenylate cyclase activity was also increased by dopamine, and the activation was not changed with or without Ca²⁺-calmodulin in fractions from the neural retina homogenate after sucrose density gradient centrifugation. The results suggest that the two regulation systems (i.e. dopamine and Ca²⁺-calmodulin) of adenylate cyclase in the rat retina appear to be independent.     

The effect of synapsin I phosphorylation upon binding of synaptic vesicles to spectrin.

We have previously demonstrated that brain spectrin is attached to small spherical synaptic vesicles via synapsin I. These studies utilized a novel microfiltration assay in which 125I-labelled synaptic vesicles were incubated with brain spectrin which was covalently attached to cellulosic membranes. In these studies purified dephosphosynapsin I was demonstrated to competitively inhibit the binding of the synaptic vesicles to the immobilized brain spectrin with a KI = 45 nM. In the current study we demonstrate that phosphorylation of synapsin I site 1 (0.74 mol Pi/mol synapsin I) with cAMP-dependent protein kinase and sites 2 and 3 (2.0 mol Pi/mol synapsin I) with Ca(2+)-calmodulin kinase II had little effect upon its interaction with brain spectrin. cAMP-dependent protein kinase phosphorylated synapsin I and Ca(2+)-calmodulin kinase II phosphorylated synapsin I both inhibited the binding of 125I-labelled synaptic vesicles to immobilized brain spectrin with a KI of 23 nM and 24 nM respectively. We conclude that phosphorylation of synapsin I does not down-regulate the interaction of synaptic vesicles with brain spectrin.     

Testosterone induces hypothalamic aromatase during early development in quail

The effects of testosterone on androgen metabolizing enzymes were examined in the developing hypothalamus of male and female quail using an in vitro radiometric assay which measures metabolite formation in individual brain samples. Testosterone (T) administered by subcutaneous silastic implants to gonadectomized 4-day-old chicks increased formation of estradiol-17 beta (E2) in both preoptic area + anterior hypothalamus (PA) and posterior hypothalamus + tuberal area (HT) to adult levels. The T-induced increase in E2 formation occurred to the same degree in both sexes. The increase was very small in control non-target areas, neostriatum intermediale + hyperstriatum ventrale (VN), of either sex. Testosterone had no effect on formation of 5 alpha-dihydrotestosterone (5 alpha-DHT), 5 beta-dihydrotestosterone (5 beta-DHT) and 5 beta-androstane 3 alpha, 17 beta-diol (5 beta, 3 alpha-diol) in PA. Kinetic analysis of the rate of E2 production by hypothalamic tissue from castrated chicks (CX-chicks) and castrates treated with T (CX + T-chicks) indicates that the increase in hypothalamic aromatase activity by T corresponds to induction of the enzyme: the Vmax (maximum velocity) was increased by T (CX-chicks, 21; CX + T-chicks, 91 fmol/mg FW/h), whereas the Km was unaffected (CX-chicks, 5.5; CX + T-chicks, 4.7 X 10(-8) M). Testosterone treatment, effective for inducing PA and HT aromatase activity, also activated crowing and caused cloacal gland development; neither of these effects were sexually dimorphic. Our results indicate that: (1) T induces aromatase specifically in the hypothalamus during early post-hatching development, other pathways of T metabolism are not affected; and (2) the inducible aromatase is not sexually dimorphic in the developing brain. Since there are sex differences in adult brain aromatase, we conclude that capacity for induction of the hypothalamic aromatase becomes sexually differentiated after the post-hatching period.     

Inhibitory effect of regucalcin on Ca(2+)-dependent protein kinase activity in rat brain cytosol: involvement of endogenous regucalcin

The effect of regucalcin, a Ca(2+)-binding protein, on Ca(2+)-dependent protein kinase activity in the brain cytosol of rats with different ages (5 and 50 weeks old) was investigated. The addition of calmodulin (10 microg/ml) or dioctanoylglycerol (5 microg/ml) in the enzyme reaction mixture caused a significant increase in protein kinase activity in the presence of CaCl2 (1 mM), indicating that Ca2+ calmodulin or protein kinase C is present in the cytosol. Such an increase was completely prevented by the addition of regucalcin (10(-7) M). Moreover, regucalcin (10(-7) M) significantly inhibited cytosolic protein kinase activity without Ca2+/calmodulin or dioctanoylglycerol addition. Meanwhile, the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the enzyme reaction mixture caused a significant elevation of protein kinase activity, suggesting an inhibitory effect of endogenous regucalcin. Brain cytosolic protein kinase activity was significantly elevated by increasing age (50-week-old rats). Also, regucalcin (10(-7) M) significantly decreased protein kinase activity without Ca(2+) addition in the brain cytosol of aged rats. However, the effect of anti-regucalcin monoclonal antibody (50 ng/ml) in elevating protein kinase activity was not seen in the brain cytosol of aged rats. These results suggest that regucalcin has an inhibitory effect on Ca(2+)-dependent protein kinase activity in rat brain cytosol, and that the effect of endogenous regucalcin may be weakened in the brain cytosol of aged rats.