The objective of this study was to examine the expression and activation of the c-kit receptor, a specific receptor for kit ligand (stem cell factor, steel factor), in rat type A spermatogonia. Testes were obtained from 9-day-old rats, decapsulated, and then subjected to sequential enzymatic digestion. The mixture of testicular cell types was then separated by sedimentation velocity at unit gravity. The isolated type A spermatogonia were characterized by light and electron microscopy. They exhibited spherical nuclei containing several nucleoli and associated chromatin clumps and organelles generally in a perinuclear location similar to that found in the in vivo 9-day-old testis. The synthesis of the c-kit receptor by the spermatogonia was established by hybridization of total RNA with a specific cDNA for mouse c-kit receptor. Two mRNA transcripts migrating at 4.8 kb and 12 kb were observed. Localization of the c-kit receptor in the isolated cells was determined by immunocytochemistry using an antibody to c-kit protein. Specific staining for c-kit receptor was observed in the cytoplasm of the isolated type A spermatogonia. Furthermore, the presence of the c-kit receptor protein in the spermatogonia was confirmed by Western blot analysis using the same antibody. The antibody recognized the c-kit receptor at approximately 160 kDa. In an attempt to determine whether this receptor has a functional significance, we examined the effect of kit ligand on the phosphorylation of the c-kit receptor. The c-kit receptor appeared to be constitutively autophosphorylated on tyrosine at low basal levels, and upon stimulation with kit ligand, the amount of phosphorylated protein increased significantly. These observations indicate that kit ligand induces autophosphorylation of the c-kit receptor, which may lead to the activation of other cellular target proteins responsible for spermatogonial proliferation and/or differentiation.

beta-amyloid (A beta), the major component of senile plaques in Alzheimer's disease (AD), normally circulates in the blood at nanomolar levels but is elevated in AD. Previous studies have found that high concentrations (10(-5)-10(-4) M) of A beta result in neuronal cell death. Here we show that physiological levels of soluble A beta can induce dysfunction in perfused rat cerebral vessels and in cultured endothelial cells. At concentrations of 10(-9)-10(-6) M, A beta induced a significant concentration-dependent reduction of NO production in endothelial cells. At 10(-8) M, A beta significantly decreased the sensitivity of cerebral vessels to acetylcholine (ACh), an endothelium dependent vasodilator. At 10(-7) M and higher concentrations, A beta significantly reduced the maximum response of vessels to ACh, and induced significant endothelial cell death. A beta (10(-9)-10(-5) M) did not cause any detectable change in nitric oxide synthase levels. The results suggest that a modest increase in the concentration of A beta above its normal physiological level in the circulation, as found in the early stages of AD, results in decreased NO production and vessel sensitivity to endothelium-dependent vasodilation that could lead to constricted blood vessels and ischemia in the surrounding tissue. Further increases in A beta concentration, which may occur in the later stages of AD, result in cell death and decreased maximum vasodilator response of cerebral vessels.

Dose-response curves were obtained from dog anterior tibial artery rings at various lengths (L) to determine whether sensitivity to norepinephrine (NE) and potassium (K+) depends on arterial circumference. The dose for half maximal response (ED50) was determined by graphical estimation and by calculation from a best fit curve. For both NE and K+: 1) ED50 was lowest (most sensitive) at L for maximum active force (Lmax) and increased significantly as L decreased from Lmax; 2) ED50 at 1.0 and 1.15 Lmax was not significantly different; 3) ED50 of repeated dose-response curves at Lmax was not significantly different; and 4) when the direction of length change was reversed (from decreasing to increasing), the direction of change in ED50 was also reversed (from increasing to decreasing). Change in the dose for 10% maximal response was the same as ED50. The results did not depend on the method of determining ED50 or on whether responses were expressed as absolute values or as relative values. The results show that sensitivity of vascular smooth muscle depends on L and that the length-sensitivity relation is similar to the length-active tension relation. Similarity of results for NE and K+ indicate that length-dependent sensitivity does not depend on the method of stimulation.

The aim of the present study was to investigate the contribution of large-conductance calcium-activated potassium (large-conductance KCa) channels to adenosine (Ado)- and nitroprusside-mediated relaxation in small coronary arteries. Canine subepicardial arteries (170 +/- 23 microns at 120 mmHg) were studied as in vitro pressurized vessels. Pressure-diameter experiments showed myogenic tone over a physiological range of pressures. Tone was increased with the thromboxane A2 analogue 9,11-dideoxy-11 alpha,9 alpha-epoxy-methanoprostaglandin F2 alpha (U-46619). Tetraethylammonium (TEA+; 1 mM) significantly inhibited Ado-induced [and by implication, adenosine 3',5'-cyclic monophosphate (cAMP)-induced] relaxations at Ado concentrations ranging from 0.1 to 10 microM with maximal inhibition (61 +/- 8%) at 1 microM Ado. The large-conductance KCa-channel blocker iberiotoxin (IbTX; 0.01-0.1 microM) inhibited Ado-mediated relaxation in a concentration-dependent manner. Inhibition by IbTX increased with increasing vessel pressure (i.e., 45 +/- 12% at 40 mmHg and 83 +/- 20% at 120 mmHg). TEA+ had a minimal effect (8 +/- 3%) on relaxation induced by nitroprusside. Similar results were found with acetylcholine and bradykinin. These results suggest that (in dog coronary arteries with diameter < 200 microns) large-conductance KCa-channel modulation may play a major role in cAMP-mediated relaxation but is not significant in guanosine 3',5'-cyclic monophosphate-mediated relaxation.

No abstract