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Human salivary gustin stimulated activity of brain calmodulin-dependent cyclic nucleotide phosphodiesterase (cAMP PDEase; 3',5'-cyclic-nucleotide phosphodiesterase, EC 3.1.4.17) in a dose-dependent manner in the absence of calmodulin. At physiological levels found in human saliva, gustin activated cAMP PDEase 5- to 6-fold. Activation of PDEase occurred with as little as 500 ng of gustin. Comparative sensitivity of activation of PDEase by gustin was intermediate between calmodulin and lysophosphatidylcholine with maximal activation and half-maximal activation (indicated in parentheses) at 3 X 10(-8) M (4.3 X 10(-9) M), 3.4 X 10(-6) M (3.4 X 10(-7) M), and 2.5 X 10(-3) M (4.0 X 10(-5) M) for calmodulin, gustin, and lysophosphatidylcholine, respectively. No other major salivary protein activated PDEase. Anticalmodulin antibody completely inhibited calmodulin-activated cAMP PDEase activity, but the antibody had no effect on gustin-activated cAMP PDEase activity. A sensitive calmodulin RIA indicated that no calmodulin was detected in any gustin preparation that activated cAMP PDEase. Both gustin and calmodulin rendered cAMP PDEase thermally labile to a similar extent and increased Vmax without affecting the apparent Km for the substrate cAMP. Activation by gustin and calmodulin was unaffected by lubrol-PX, trypsin inhibitor, pepstatin A, or leupeptin. In the presence of 1 mM EGTA, gustin activated cAMP PDE 5- to 6-fold, but the activating ability was completely lost after gustin was heated at 100 degrees C for 5 min. In contrast, calmodulin lost all activating ability in the presence of 1 mM EGTA, whereas heating calmodulin at 100 degrees C for 5 min did not affect its activation of cAMP PDEase. Lysophosphatidylcholine-activation of cAMP PDEase, like gustin activation, was unaffected by EGTA, but lysophosphatidylcholine-activation of cAMP PDEase, like calmodulin activation, was unaffected by heating at 100 degrees C for 5 min.

Two groups of male Sprague-Dawley rats, one fed zinc-deficient diet, ad libitum, the other, pair-fed with the same diet, but given supplemental zinc in the drinking water (8 mg Zn++/ml) were studied. After ten weeks of diet, rats were exsanguinated and zinc and calmodulin concentrations in brain and testis were measured. Mean zinc concentration in testis was significantly decreased in rats fed zinc-deficient diet without supplemental Zn++, but mean zinc concentration in brain was not different. Similarly, mean calmodulin concentration in testis was decreased in rats fed zinc-deficient diet without supplemental Zn++ whereas mean calmodulin concentration in brain was not different. Distribution studies of zinc and calmodulin showed that both zinc and calmodulin were released more freely into soluble fractions of testis in rats fed zinc-deficient diet without supplemental Zn++. These results indicate, for the first time in in vivo studies, that zinc influences the calmodulin content of testis.

Calmodulin is higher in particulate fractions from bovine taste buds containing taste bud membranes which specifically bind sweet tastants compared to corresponding fractions from control non-taste bud bearing lingual epithelial tissue. As biochemical purity (i.e., membrane enzyme marker activity) of these membrane enriched fractions increased (P4B greater than P3B greater than P2B) calmodulin correspondingly increased (P4B greater than P3B greater than P2B); these increases also correlated with increased membrane purity as demonstrated by electron microscopy. All PB subfractions from taste buds contained a greater membrane concentration than those from PD subfractions and calmodulin was significantly increased in each corresponding subfraction. The presence of calmodulin in taste bud membranes, its correlation with membrane purification and reports that numerous drugs which induce taste loss are potent inhibitors of calmodulin suggest a role for calmodulin in taste function.

Plasma-membrane-enriched fractions of canine aortic smooth muscle possess an ATP-supported Ca2+ accumulation which has an absolute requirement for Mg2+ and a high affinity for Ca2+ (Km approximately 0.5 microM). The rate of ATP-supported Ca2+ transport is not affected by several calmodulin antagonists, but is stimulated by exogenously added calmodulin. The maximal effect of calmodulin on the rate of ATP-dependent Ca2+ transport (at 5.0 microM Ca2+) occurs at 10 micrograms/ml calmodulin and represents an approximately 3-fold stimulation. This calmodulin stimulation of Ca2+ transport does not require pretreatment of the membranes by EGTA and is an intrinsic property of the plasma membranes. A high-affinity Ca2+-ATPase (Km for Ca2+ approximately 0.5 microM) is also present in the aortic smooth muscle plasma membrane. This high-affinity Ca2+-ATPase does not require Mg2+ for catalytic activity, but is in fact inhibited by increasing Mg2+ concentrations. Calmodulin at concentrations effective for the stimulation of the ATP-dependent Ca2+ transport has no effect on the high-affinity Ca2+-ATPase activity or on the basal ATPase activity stimulated by 5 mM Mg2+ or Ca2+. Our results indicate that isolated plasma membranes of canine aortic smooth muscle contain no endogenous calmodulin. The ability of exogenously added calmodulin to stimulate the rate of ATP-dependent Ca2+ transport by vascular smooth muscle plasma membranes suggests that calmodulin may play a role in lowering the cytoplasmic concentration of ionized calcium during vasodilatation. An Mg2+-independent, but not an Mg2+-dependent high-affinity Ca2+-ATPase, was identified in the plasma membranes. This may be separate from the plasmalemmal Ca2+-pump.

Parotid saliva calmodulin was found both in 32 normal volunteers and in 60 patients with taste and smell dysfunction; salivary calmodulin concentration was significantly lower in the patients than in the volunteers. There were no differences in salivary calmodulin concentration with respect to age, sex, or salivary flow rate in either normal volunteers or patients. When patients were categorized by diagnosis, calmodulin concentration was found to be decreased in all patient groups. The concentration of calmodulin in saliva was about 10 times that found in serum, suggesting that the parotid gland is a major source of this protein.

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.