A radioisotope flux-rapid-quench-Millipore filtration method is described for determining the effects of Ca2+, adenine nucleotides, and Mg2+ on the Ca2+ release behaviour of "heavy" sarcoplasmic reticulum (SR) vesicles. Rapid 45Ca2+ efflux from passively loaded vesicles was blocked by the addition of Mg2+ and ruthenium red. At pH 7 and 10(-9) M Ca2+, vesicles released 45Ca2+ with a low rate (k = 0.1 s-1). An increase in external Ca2+ concentration to 4 microM or the addition of 5 mM ATP or the ATP analogue adenosine 5'-(beta,gamma-methylenetriphosphate) (AMP-PCP) resulted in intermediate 45Ca2+ release rates. The maximal release rate was observed in media containing 4 microM Ca2+ and 5 mM AMP-PCP and had a first-order rate constant of 30-100 s-1. Mg2+ partially inhibited Ca2+- and nucleotide-induced 45Ca2+ efflux. In the absence of AMP-PCP, 45Ca2+ release was fully inhibited at 5 mM Mg2+ or 5 mM Ca2+. The composition of the release media was systematically varied, and the flux data were expressed in the form of Hill equations. The apparent n values of activation of Ca2+ release by ATP and AMP-PCP were 1.6-1.9. The Hill coefficient of Ca2+ activation (n = 0.8-2.1) was dependent on nucleotide and Mg2+ concentrations, whereas the one of Mg2+ inhibition (n = 1.1-1.6) varied with external Ca2+ concentration. These results suggest that heavy SR vesicles contain a "Ca2+ release channel" which is capable of conducting Ca2+ at rates comparable with those found in intact muscle. Ca2+, AMP-PCP (ATP), and Mg2+ appear to act at noninteracting or interacting sites of the channel.

To examine the possible role of microtubule-based transport in testicular function, we used immunofluorescent techniques to study the presence and localization of the microtubule mechanoenzymes cytoplasmic dynein (a slow-growing end-directed motor) and kinesin (a fast-growing end-directed motor) within rat testis. Cytoplasmic dynein immunofluorescence was observed in Sertoli cells during all stages of spermatogenesis, with a peak in apical cytoplasm during stages IX-XIV. Cytoplasmic dynein immunofluorescence was also localized within Sertoli cells to steps 9-14 (stages IX-XIV) germ cell-associated ectoplasmic specializations. In germ cells, cytoplasmic dynein immunofluorescence was observed in manchettes of steps 15-17 (stages I-IV) spermatids, and small, hollow circular structures were seen in the cytoplasm of step 17 and step 18 spermatids during stages V and VI. Kinesin immunofluorescence was observed in manchettes of steps 10-18 spermatids (stages X-VI). The stage-dependent apical Sertoli cell cytoplasmic dynein immunofluorescence, in conjunction with the previously reported orientation of Sertoli cell microtubules (slow-growing ends toward the lumen) and peak secretion of androgen-binding protein and transferrin, is consistent with the hypothesis that cytoplasmic dynein is involved in Sertoli cell protein transport and secretion. Further, the localization of cytoplasmic dynein and kinesin to manchettes is consistent with current hypotheses concerning manchette function.

The seminiferous tubule of the testis contains a rich variety of microtubule networks and of microtubule-associated proteins (MAPs). Tau is a heat-stable MAP previously believed to be limited in its expression in mammals to the nervous system. We have identified tau in rat and bovine testis, a unique non-neuronal location, using biochemical, molecular, and immunologic approaches. SDS-PAGE of ammonium sulfate-fractionated, testis heat-stable MAPs resulted in an enrichment of bands that comigrated with rat brain tau. Only the 35-45% precipitated ammonium sulfate fraction induced microtubule assembly. Immunoblotting with monoclonal anti-tau antibodies demonstrated tau immunoreactivity in these testis MAP preparations. Northern analysis of total rat testis RNA demonstrated a 1.7-kb band that hybridized with a 51-nucleotide oligomer complementary to a conserved portion of the tau transcript. This 51-mer identified a similar 1.7-kb minor band and an additional 6-kb major band in Northern analysis of total rat brain RNA. Finally, in the bull testis, immunohistochemistry localized tau to the spermatid manchette, a transient, cross-linked microtubule network of unknown function. As spermatid elongation begins, the manchette forms a sheath around the posterior aspect of the nucleus, but, by the completion of nuclear condensation, the manchette is largely disassembled. Tau most likely plays a structural role in the manchette; however, tau immunoreactivity also was observed in late stage I spermatids prior to manchette formation, suggesting that tau may serve a function in manchette assembly.

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Administration of meso‐tetra(4‐sulfonatophenyI)porphine (TPPS4) to rats resulted in cyto‐skeletal abnormalities with loss of microtubules in unmyelinated peripheral nerve axons (Winkelman and Collins, 1987). That in vivo toxic effect prompted the present in vitro study of microtubule assembly inhibition by porphyrins and related compounds. Several approaches were used to measure microtubule assembly inhibition including: (1) determination of the final optical density at 350 nm, (2) analysis of supernatant unassembled tubulin by direct measurement and by colchicine binding, and (3) ultrastructural examination for the presence of typical microtubule profiles. Of the 11 compounds studied, TPPS4, two less sulfonated TPPS congeners and me.so‐tetra(N′‐methyl‐4‐pyridyl)porphine caused profound microtubule assembly inhibition. Afoo‐tetra(4‐carboxyphenyl)porphinc produced an intermediate level of microtubule assembly inhibition. The remaining compounds, including hemato‐porphyrin D (HpD), had little or no effect upon microtubule assembly. In phytohemagglutinin stimulated lymphocytes, nonphotoactivated TPPS4 produced metaphase arrest to the same extent as colcemid. Thus, the specific interaction of TPPS4 with tubulin produces microtubule assembly inhibition in vitro and metaphase arrest in cell culture. Tubulin binding could explain the intracellular concentration of TPPS4 and TPPS‐like compounds, and differences in the biological behavior of these from other porphyrins.

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