Protein kinase A and protein kinase C(alpha)/PPP1CC2 play opposing roles in the regulation of phosphatidylinositol 3-kinase activation in bovine sperm.

In order to acquire fertilization competence, spermatozoa have to undergo biochemical changes in the female reproductive tract, known as capacitation. Signaling pathways that take place during the capacitation process are much investigated issue. However, the role and regulation of phosphatidylinositol 3-kinase (PI3K) in this process are still not clear. Previously, we reported that short-time activation of protein kinase A (PRKA, PKA) leads to PI3K activation and protein kinase C(alpha)(PRKCA, PKC(alpha)) inhibition. In the present study, we found that during the capacitation PI3K phosphorylation/activation increases. PI3K activation was PRKA dependent, and down-regulated by PRKCA. PRKCA is found to be highly active at the beginning of the capacitation, conditions in which PI3K is not active. Moreover, inhibition of PRKCA causes significant activation of PI3K. Similar activation of PI3K is seen when the phosphatase PPP1 is blocked suggesting that PPP1 regulates PI3K activity. We found that during the capacitation PRKCA and PPP1CC2 (PP1gamma2) form a complex, and the two enzymes were degraded during the capacitation, suggesting that this degradation enables the activation of PI3K. This degradation is mediated by PRKA, indicating that in addition to the direct activation of PI3K by PRKA, this kinase can enhance PI3K phosphorylation indirectly by enhancing the degradation and inactivation of PRKCA and PPP1CC2.

Experimental verocytotoxemia in rabbits.

The clinicopathologic effects of intravenously administered purified verocytotoxin 1 (VT1; Shiga-like toxin 1) in 2-kg male rabbits was studied. The 50% lethal dose was 0.2 micrograms of protein per kg of body weight (2 x 10(4) 50% cytotoxic doses per kg). The clinical features included nonbloody diarrhea and a progressive flaccid paresis, usually culminating in death. The histopathology was characterized by edema and hemorrhage in the mucosa and submucosa of the cecum and edema, hemorrhage, and neuronal necrosis in the brain and gray matter of the spinal cord. Thrombotic microangiopathy, the characteristic histopathologic renal lesion in the hemolytic-uremic syndrome, was also found to be the underlying lesion in verocytotoxemic rabbits. To determine the specific distribution of VT1 in rabbit tissues, purified 125I-labelled VT1 was administered intravenously to 20 rabbits (both immunologically naive and VT1-immune rabbits). The highest specific uptake of 125I-VT1 was in the spinal cord, brain, cecum, colon, and small bowel in unimmunized animals but in the liver, spleen, and lungs in immune animals. Immunofluorescent staining of cecal and spinal cord tissues after intravenous administration of VT1 showed evidence of specific vascular endothelial cell binding of the toxin. The striking correlation of the central nervous system and gastrointestinal localization of 125I-VT1 with the sites of known histopathology is consistent with direct toxin-mediated injury to these tissues, initiated by the specific binding of VT1 to the vascular endothelium. We conclude that the vascular damage induced by VT1 in affected rabbit tissues is similar to that seen in the kidneys and other tissues in patients with verocytotoxin-producing Escherichia coli-associated hemolytic-uremic syndrome. This suggests that although the rabbit model fails to replicate human hemolytic-uremic syndrome, it is useful for studying the pathogenesis of the vascular lesions in verocytotoxin-producing E. coli-associated diseases.