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Anesthetic treatment has been associated with widespread apoptotic neurodegeneration in the neonatal rodent brain. It has recently been suggested that propofol, a short-acting intravenous anesthetic agent, may have a potential as a neuroprotective agent. An apoptotic pathway mediated through endoplasmic reticulum (ER) stress has been attracting attention. ER stress is associated with accumulation of unfolded or misfolded proteins in ER, and ER stress-induced apoptosis is implicated in a wide range of diseases, including ischemia/reperfusion injury, neurodegeneration, and diabetes. We investigated whether thapsigargin-induced ER stress is prevented by propofol in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were pretreated with various concentrations of propofol (1-10 μM) for 3h before co-treatment with 0.5 μM thapsigargin and propofol for 20 h. Levels of ssDNA, specific evidence of apoptosis, and biomarkers of ER stress (mRNA expression of Chop and sXbp-1) were determined. We also assayed calpain and caspase-4 activities and intracellular Ca(2+) ([Ca(2+)]i) levels. Thapsigargin-induced increases in ssDNA levels, expressions of ER stress biomarkers, activities of caspase-4 and calpain, and level of [Ca(2+)]i were suppressed by co-incubation with propofol. Our data indicate the possibility that propofol inhibits the Ca(2+) release from ER at clinically employed dose levels. These results demonstrate that propofol suppresses the ER stress-induced apoptosis in this cell system, and may have the neuroprotective potency. It may also be a promising agent for preventing damage from cerebral ischemia or edema.

The effects of intracellular Cl−concentration ([Cl−]i) on acetylcholine (ACh)-stimulated exocytosis were studied in guinea pig antral mucous cells by video microscopy. ACh activated Ca2+-regulated exocytosis (an initial phase followed by a sustained phase). Bumetanide (20 μM) or a Cl−-free (NO3−) solution enhanced it; in contrast, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a Cl−channel blocker) decreased it and eliminated the enhancement induced by bumetanide or NO3−solution. ACh and Ca2+dose-response studies demonstrated that NO3−solution does not shift their dose-response curves, and ATP depletion studies by dinitrophenol or anoxia demonstrated that exposure of NO3−solution prior to ATP depletion induced an enhanced initial phase followed by a sustained phase, whereas exposure of NO3−solution after ATP depletion induced only a sustained phase. Intracellular Ca2+concentration ([Ca2+]i) measurements showed that bumetanide and NO3−solution enhanced the ACh-stimulated [Ca2+]iincrease. Measurements of [Cl−]irevealed that ACh decreases [Cl−]iand that bumetanide and NO3−solution decreased [Cl−]iand enhanced the ACh-evoked [Cl−]idecrease; in contrast, NPPB increased [Cl−]iand inhibited the [Cl−]idecrease induced by ACh, bumetanide, or NO3−solution. These suggest that [Cl−]imodulates [Ca2+]iincrease and ATP-dependent priming. In conclusion, a decrease in [Cl−]iaccelerates ATP-dependent priming and [Ca2+]iincrease, which enhance Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells.

Ca2+-regulated exocytosis is enhanced by an autocrine mechanism via the PGE2–cAMP pathway in antral mucous cells of guinea-pigs. The inhibition of the PGE2–cAMP pathway by H-89 (an inhibitor of protein kinase A, PKA) or aspirin (ASA, an inhibitor of cyclo-oxygenase, COX) decreased the frequency of ACh-stimulated exocytotic events by 60%. Indomethacin (IDM, an inhibitor of COX), however, decreased the frequency of ACh-stimulated exocytotic events only by 30%. Moreover, IDM increased the frequency of ACh-stimulated exocytotic events by 50% in H-89-treated or ASA-treated cells. IDM inhibits the synthesis of Prostaglandin (PGG/H) and (15R)-15-hydroxy-5,8,11 cis-13-trans-eicosatetraenoic acid (15R-HPETE), while ASA inhibits only the synthesis of PGG/H. Thus, IDM may accumulate arachidonic acid (AA). AACOCF3 or N-(p-amylcinnamoyl) anthranilic acid (ACA; both inhibitors of phospholipase A2, PLA2), which inhibits AA synthesis, decreased the frequency of ACh-stimulated exocytotic events by 60%. IDM, however, did not increase the frequency in AACOCF3-treated cells. AA increased the frequency of ACh-stimulated exocytotic events in AACOCF3- or ASA-treated cells, similar to IDM in ASA- and H-89-treated cells. Moreover, in the presence of AA, IDM did not increase the frequency of ACh-stimulated exocytotic events in ASA-treated cells. The PGE2 release from antral mucosa indicates that inhibition of PLA2 by ACA inhibits the AA accumulation in unstimulated and ACh-stimulated antral mucosa. The dose–response study of AA and IDM demonstrated that the concentration of intracellular AA accumulated by IDM is less than 100 nm. In conclusion, IDM modulates the ACh-stimulated exocytosis via AA accumulation in antral mucous cells.

The effects of intracellular Ca2+ concentration, [Ca2+]i, on the volume of rat alveolar type II cells (AT-II cells) were examined. Perfusion with a Ca2+-free solution induced shrinkage of the AT-II cell volume in the absence or presence of amiloride (1 μm, an inhibitor of Na+ channels); however, it did not in the presence of 5-(N-methyl-N-isobutyl)-amiloride (MIA, an inhibitor of Na+–H+ exchange). MIA decreased the volume of AT-II cells. Inhibitors of Cl−–HCO3− exchange, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS) also decreased the volume of AT-II cells. This indicates that the cell shrinkage induced by a Ca2+-free solution is caused by a decrease in NaCl influx via Na+–H+ exchange and Cl−–HCO3− exchange. Addition of ionomycin (1 μm), in contrast, induced cell swelling when AT-II cells were pretreated with quinine and amiloride. This swelling of the AT-II cells is not detected in the presence of MIA. Intracellular pH (pHi) measurements demonstrated that the Ca2+-free solution or MIA decreases pHi, and that ionomycin increases it. Ionomycin stimulated the pHi recovery after an acid loading (NH4+ pulse method), which was not noted in MIA-treated AT-II cells. Ionomycin increased [Ca2+]i in fura-2-loaded AT-II cells. In conclusion, the Na+–H+ exchange activities of AT-II cells, which maintain the volume and pHi, are regulated by [Ca2+]i.

Terbutaline (10 microm) induced a triphasic volume change in alveolar type II (AT-II) cells: an initial shrinkage (initial phase) followed by cell swelling (second phase) and a gradual shrinkage (third phase). The present study demonstrated that the initial and the third phases are evoked by the activation of K+ and Cl- channels and the second phase is evoked by the activation of Na+ and Cl- channels. Ouabain blocked the third phase, although it did not block the initial and second phases. This suggests that the third phase is triggered by the Na+-K+ pump. Tetraethylammonium (TEA, a K+ channel blocker) decreased the volume of AT-II cells and enhanced the terbutaline-stimulated third phase, although quinidine, another K+ channel blocker, increased the volume of AT-II cells. The TEA-induced cell shrinkage was inhibited by ouabain, suggesting that TEA increases Na+-K+ pump activity. Ba2+, 2,3-diaminopyridine and a high [K+]o (30 mm) similarly decreased the volume of AT-II cells. These findings suggest that depolarization induced by TEA increases Na+-K+ pump activity, which increases [K+]i. This [K+]i increase, in turn, hyperpolarizes membrane potential. Valinomycin (a K+ ionophore), which induces hyperpolarization, decreased the volume of AT-II cells and enhanced the third phase in these cells. In conclusion, in terbutaline-stimulated AT-II cells, an increase in Na+-K+ pump activity hyperpolarizes the membrane potential and triggers the third phase by switching net ion transport from NaCl entry to KCl release.

A cross-sectional study of Japanese female commercial sex workers (FCSWs) working in massage parlors with cell baths (MPCBs) was conducted between July 1999 and December 2001. The study subjects were 171 FCSWs aged from 19 to 36 years. A questionnaire included sexual characteristics in addition to working name and date of birth. We serologically or bacteriologically confirmed the prevalence of HIV-1, HIV-2, hepatitis B virus (HBV), hepatitis C virus (HCV), Chlamydia trachomatis, Neisseria gonorrhoeae, syphilis, and trichomoniasis. There were no differences in the clinical characteristics of FCSWs working in standard-class MPCBs (group A) and those working in expensive-class MPCBs (group B). With respect to sexual characteristics, HIV-1 and HIV-2 were not confirmed in any subjects, but N. gonorrhoeae was detected in 1.2%. Use of condoms was 98.4% in group A and 83.3% in group B (P < 0.01). No HIV infection and an extremely low prevalence of sexually transmitted diseases (STDs) were recognized in Japanese FCSWs working in standard- and expensive-class MPCBs.

In this study, we examined whether the transmission of Helicobacter pylori occurred in married couples. We also examined whether a correlation existed between H. pylori seropositivity in a parent and an offspring, which may be related to H. pylori transmission. A cross-sectional study of residents living in a rural area in Japan was conducted in 1998. The study population consisted of 1910 volunteers, aged from 22 to 79 years, residing in this area. We serologically confirmed the presence of the antigen, CagA (cytotoxin-associated gene A), of H. pylori, using a recombinant CagA antigen, in addition to examining for antibodies to H. pylori, as determined by an IgG-specific enzyme immunoassay. The data were analyzed using logistic regression models. A significant association of H. pylori seropositivity was observed (P < 0.001) in married couples, but no significant difference with respect to the presence of anti-CagA antibodies was observed in the married couples with H. pylori seropositive results (P = 0.053). The odds ratio was 8.08 (95% confidence interval [CI], 1.39–40.05) for infection in offspring with seropositive mothers and 2.93 (95% CI, 0.25–33.85) for infection in offspring with seropositive fathers when compared with seronegative fathers or mothers as the reference. There was a concordance between the presence or absence of anti-CagA antibodies in 11 of 13 groups of mothers and offspring (84.6%) compared with concordance in 6 of 14 groups of fathers and offspring (42.9%; P < 0.05). Our results suggest that maternal influence is likely to be more powerful than paternal influence in the transmission of H. pylori.

Neurocalcin (NC), a neuron-specific EF-hand Ca2+-binding protein, purified from bovine brain [Terasawa, Nakano, Kobayashi and Hidaka (1992) J. Biol. Chem. 267, 19596-19599] contains multiple isoforms. We previously cloned NCdelta from bovine brain and showed high expression in neuronal tissues [Okazaki, Watanabe, Ando, Hagiwara, Terasawa and Hidaka (1992) Biochem. Biophys. Res. Commun. 185, 147-153]. We report here the molecular cloning and expression of a cDNA encoding bovine brain NCalpha. The translated bovine protein is 191 amino acids long and shares 69.1% of its amino acid sequence with NCdelta. Recombinant NCalpha migrates as a single 23 kDa band and exhibits a Ca2+-dependent mobility shift on SDS/PAGE. Analysis of fluorescence emission spectra showed the Ca2+-induced peak at 337 nm. Interestingly, the mobility shift and the fluorescence intensity at 337 nm were larger for NCalpha than for NCdelta. In Ca2+-overlay experiments, however, the apparent affinity of NCalpha for 45Ca2+ was similar to that of NCdelta. Immunohistochemical analysis revealed NCalpha expression in the granular layer of the rat cerebellar cortex whereas NCdelta was found in the Purkinje cell layer. In the rat olfactory bulb, NCalpha was located in external tufted cells, and NCdelta was found in the periglomerular cells. These data demonstrate that NC isoforms differ in their tissue distribution and conformational changes induced by Ca2+ binding. Thus differential regulation of the two NC isoforms may be involved in control of neuron function.

Distribution of neurocalcin, a calcium-binding protein having three EF hand motifs, in the rat sensory ganglia was demonstrated immunochemically and immunohistochemically. Immunoblot analysis of trigeminal, nodose and dorsal root ganglia homogenates revealed an immunoreactive band at approximately 24 kDa. Neurons labeled by the neurocalcin-antiserum represent 54%, 41% and 46% cells in the trigeminal, nodose and dorsal root ganglia, respectively. Size distribution of immunopositive cells showed a varying range. Most large cells (more than 80%) showed immunoreactivity in the trigeminal and dorsal root ganglia. A double immunofluorescent study was performed to determine the colocalization with calbindin D28k or parvalbumin, which are both calcium-binding proteins. In the trigeminal and dorsal root ganglia, almost all calbindin- or parvalbumin-immunoreactive neurons showed neurocalcin-immunoreactivity, whereas approximately 30-40% neurocalcin-immunopositive cells had calbindin- or parvalbumin-immunoreactivity. In the nodose ganglia, parvalbumin showed localization similar to other ganglia, but about half the calbindin-immunoreactive neurons had neurocalcin-immunoreactivity. These studies show that neurocalcin-immunopositive neurons are widely distributed in the sensory ganglia and most calbindin- or parvalbumin-immunoreactive neurons also contain neurocalcin. In the sensory neurons, neurocalcin may have a significant role in calcium signaling.

Concentrations of estradiol (E), testosterone (T), and progesterone (P) and levels of the 1.9-kb and 4.0-kb mRNA encoding P450 17 alpha-hydroxylase (P450 17 alpha) and P450aromatase (P450arom), respectively, were measured in the isolated granulosa, theca interna, and theca externa of the largest preovulatory (F1-F4) follicles of the chicken 20 h and 3 h before expected ovulation of F1. In the granulosa layer, E and T concentrations remained low throughout this period of follicular growth, whereas P concentrations were high 3 h before ovulation in F2 and F1 follicles. E levels were highest in the theca interna and externa of the smallest (F4) follicle, decreasing progressively with follicular growth. T content of theca interna and granulosa was greater in less mature F4-F2 follicles than in the most mature (F1) follicle, whereas theca externa contained little T. P was quantifiable in significant amounts only in granulosa tissue, peaking in F2 and F1 follicles at 3 h before ovulation, at which time plasma levels of steroids also rose significantly. P450 17 alpha was expressed mainly in theca interna, whereas the P450arom mRNA levels found in theca externa reflected the pattern of E in this compartment. Expression of these two genes was very low in granulosa tissue, remaining unchanged during follicular growth. These results indicate that E and T synthesis in the chicken ovary is regulated, at least in part, at mRNA levels of P450 17 alpha and P450arom in the theca interna and theca externa layers during follicular growth.

Ca2+-regulated exocytosis is enhanced by an autocrine mechanism via the PGE2-cAMP pathway in antral mucous cells of guinea-pigs. The inhibition of the PGE2-cAMP pathway by H-89 (an inhibitor of protein kinase A, PKA) or aspirin (ASA, an inhibitor of cyclo-oxygenase, COX) decreased the frequency of ACh-stimulated exocytotic events by 60%. Indomethacin (IDM, an inhibitor of COX), however, decreased the frequency of ACh-stimulated exocytotic events only by 30%. Moreover, IDM increased the frequency of ACh-stimulated exocytotic events by 50% in H-89-treated or ASA-treated cells. IDM inhibits the synthesis of Prostaglandin (PGG/H) and (15R)-15-hydroxy-5,8,11 cis-13-trans-eicosatetraenoic acid (15R-HPETE), while ASA inhibits only the synthesis of PGG/H. Thus, IDM may accumulate arachidonic acid (AA). AACOCF3 or N-(p-amylcinnamoyl) anthranilic acid (ACA; both inhibitors of phospholipase A2, PLA2), which inhibits AA synthesis, decreased the frequency of ACh-stimulated exocytotic events by 60%. IDM, however, did not increase the frequency in AACOCF3-treated cells. AA increased the frequency of ACh-stimulated exocytotic events in AACOCF3- or ASA-treated cells, similar to IDM in ASA- and H-89-treated cells. Moreover, in the presence of AA, IDM did not increase the frequency of ACh-stimulated exocytotic events in ASA-treated cells. The PGE2 release from antral mucosa indicates that inhibition of PLA2 by ACA inhibits the AA accumulation in unstimulated and ACh-stimulated antral mucosa. The dose-response study of AA and IDM demonstrated that the concentration of intracellular AA accumulated by IDM is less than 100 nm. In conclusion, IDM modulates the ACh-stimulated exocytosis via AA accumulation in antral mucous cells.

Summary Objectives To elucidate risk factors for acute coronary syndromes (ACS), the present study examined whether Helicobacter pylori infection is a risk factor for patients with ACS. Methods We studied 33 male patients with acute coronary syndromes (ACS). All patients were incidence cases of ACS that they did not have a past history of IHD and were at the first onset of ACS. A control group was consisted of 66 males. Controls were at random selected from outpatients. All controls had normal resting electrocardiogram and had no history of IHD. H. pylori seropositivity was determined by an IgG-specific enzyme linked immunosorbent assay (ELISA). We serologically confirmed the presence of antibodies specific to the antigen CagA of H. pylori , using CagA ELISA. Results Seropositive rate of IgG antibodies in patients with ACS was 87.9%. A rate of in controls was 66.7%. After adjustment for age, a statistically significant association was found in H. pylori seropositivity between ACS and controls (OR, 3.74; 95% CI, 1.15–12.13). This relation was also significant after adjusted for potential confounding factors (OR, 4.09; 95% CI, 1.10–15.17). Anti-CagA positive H. pylori were significantly recognized in ACS (adjusted OR, 3.58; 95% CI, 1.08–11.82). However, this significant association was disappeared after adjusted for potential confounding factors ( P =0.054). Conclusions We confirmed a significant link between H. pylori infection and ACS. H. pylori infection is likely to be a risk factor for ACS.

The effects of intracellular Ca2+ concentration, [Ca2+]i, on the volume of rat alveolar type II cells (AT-II cells) were examined. Perfusion with a Ca2+-free solution induced shrinkage of the AT-II cell volume in the absence or presence of amiloride (1 microm, an inhibitor of Na+ channels); however, it did not in the presence of 5-(N-methyl-N-isobutyl)-amiloride (MIA, an inhibitor of Na+-H+ exchange). MIA decreased the volume of AT-II cells. Inhibitors of Cl(-)-HCO3- exchange, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) also decreased the volume of AT-II cells. This indicates that the cell shrinkage induced by a Ca2+-free solution is caused by a decrease in NaCl influx via Na+-H+ exchange and Cl(-)-HCO3- exchange. Addition of ionomycin (1 microm), in contrast, induced cell swelling when AT-II cells were pretreated with quinine and amiloride. This swelling of the AT-II cells is not detected in the presence of MIA. Intracellular pH (pHi) measurements demonstrated that the Ca2+-free solution or MIA decreases pHi, and that ionomycin increases it. Ionomycin stimulated the pHi recovery after an acid loading (NH4+ pulse method), which was not noted in MIA-treated AT-II cells. Ionomycin increased [Ca2+]i in fura-2-loaded AT-II cells. In conclusion, the Na+-H+ exchange activities of AT-II cells, which maintain the volume and pHi, are regulated by [Ca2+]i.

Terbutaline (10 microm) induced a triphasic volume change in alveolar type II (AT-II) cells: an initial shrinkage (initial phase) followed by cell swelling (second phase) and a gradual shrinkage (third phase). The present study demonstrated that the initial and the third phases are evoked by the activation of K+ and Cl- channels and the second phase is evoked by the activation of Na+ and Cl- channels. Ouabain blocked the third phase, although it did not block the initial and second phases. This suggests that the third phase is triggered by the Na+-K+ pump. Tetraethylammonium (TEA, a K+ channel blocker) decreased the volume of AT-II cells and enhanced the terbutaline-stimulated third phase, although quinidine, another K+ channel blocker, increased the volume of AT-II cells. The TEA-induced cell shrinkage was inhibited by ouabain, suggesting that TEA increases Na+-K+ pump activity. Ba2+, 2,3-diaminopyridine and a high [K+]o (30 mm) similarly decreased the volume of AT-II cells. These findings suggest that depolarization induced by TEA increases Na+-K+ pump activity, which increases [K+]i. This [K+]i increase, in turn, hyperpolarizes membrane potential. Valinomycin (a K+ ionophore), which induces hyperpolarization, decreased the volume of AT-II cells and enhanced the third phase in these cells. In conclusion, in terbutaline-stimulated AT-II cells, an increase in Na+-K+ pump activity hyperpolarizes the membrane potential and triggers the third phase by switching net ion transport from NaCl entry to KCl release.

A cross-sectional study among Japanese female commercial sex workers (CSWs) working in soaplands (massage parlours with baths) was conducted between July 1999 and June 2000. The study subjects included 135 CSWs aged from 20 to 36 years. A questionnaire included sexual characteristics in addition to real name, working name, and date of birth. We serologically or bacteriologically confirmed prevalences of HIV-1, HIV-2, hepatitis B virus (HBV), hepatitis C virus (HCV), Chlamydia trachomatis, Neisseria gonorrhoeae, syphilis, and trichomonads. Anti-HCV positive was 0.7%. Although anti-HBs positive was 15.6% no HBsAg was confirmed. Seroprevalence of C. trachomatis was 8.9% and of syphilis was 4.4%. The other STDs studied were not detected. A statistically significant difference was observed between middle-class CSWs and high-class ones about condom use ( P < 0.01). Prevalences of STDs among Japanese female CSWs working in middle- and high-class soaplands were evidently lower than those reported previously.

HF-2035, 2-[N-(2-aminoethyl)-N-(2,4,5-trichlorobenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, was synthesized and its effects on calmodulin-dependent enzymes were investigated. HF-2035 inhibited calmodulin kinase I, calmodulin kinase II and myosin light-chain kinase with IC50 values of 1.3 microM, 1.6 microM and 68 microM, respectively. HF-2035 also inhibited the activity of recombinant rat neuronal nitric oxide synthase, one of the calmodulin-dependent enzymes, with a Ki of 0.78 microM. Partially purified nitric oxide synthase of rat brain was also inhibited by HF-2035 with an IC50 of 3.2 microM. Kinetic analysis indicated that this inhibitory effect of HF-2035 was competitive with respect to calmodulin. We examined the effects of HF-2035 on constitutive nitric oxide synthase in a bioassay using vascular strips of rabbit carotid artery with and without endothelium. HF-2035 inhibited acetylcholine- and calcium ionophore, A23187 (6S-[6 alpha (2S*,3S*),8 beta (R*),9 beta, 11 alpha]-5- (methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)- ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazol ecarboxylic acid)-induced relaxation of endothelium-intact strips with an ED50 of 1.5 +/- 0.5 microM and 2.8 +/- 1 microM, respectively. This compound, however, did not inhibit N-nitroso-N-morpholinoaminoacetonitrile (SIN-1A), an exogenous nitric oxide donor, -induced relaxation of endothelium-denuded strips. W-7 (N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide) inhibited acetylcholine-induced relaxation with an ED50 of 46 +/- 7 microM, which was 30-fold less potent than HF-2035. HF-2035 was unable to inhibit the activity of the inducible form of nitric oxide synthase in isolated thoracic aorta of rat treated with Escherichia coli lipopolysaccharide. These findings suggest that HF-2035 is a new and potent calmodulin antagonist, and may be used as a mother compound to develop more selective inhibitors of constitutive nitric oxide synthase.

We surveyed rabbit brain cytosol for a new Ca2+/calmodulin (CaM)-dependent kinase. The renaturation blotting assay (RBA) exploits the ability of blotted SDS-denatured proteins to regain enzymic activity after guanidine treatment. Using RBA, we found that the eluate of rabbit brain cytosol from a CaM affinity column contains at least four electrophoretically distinct protein kinase bands which were autophosphorylated in a Ca2+/CaM-dependent manner. The 49 kDa band and the 60 kDa band were alpha and beta subunit of CaM kinase II, and the 42 kDa band was presumed to be CaM kinase I, but the 80 kDa band could not be attributed to any reported Ca2+/CaM-dependent protein kinases. The 80 kDa protein kinase was isolated by three-step chromatography. We examined the phosphorylation of exogenous substrates by 80 kDa protein kinase, and histone IIIs and myosin light chain were phosphorylated in a Ca2+/CaM-dependent manner. W-7, a specific inhibitor for calmodulin, inhibited this kinase activity, but KN-62, a specific inhibitor for CaM kinase II, had no effect on this protein kinase activity. Autoradiography using boiled rabbit brain homogenate as substrate showed three intrinsic substrates (80 kDa, 60 kDa and 42 kDa), which were phosphorylated in a Ca2+/CaM-dependent manner. These findings suggest that a new Ca2+/CaM-dependent protein kinase could be identified by the RBA.