Corpus cavernosum smooth muscle (CCSM) from rabbits made diabetic for 6 months as a result of alloxan injection exhibited increased sensitivity (3vs 9 nM EC 50 ) and generated 20-50% greater force to endothelin-1 (ET-1) compared to CCSM from normal rabbits. In contrast, the force produced by the CCSM in response to KCl and phenylephrine was not significantly altered in diabetic CCSM. The increased ET-1 sensitivity is associated with a two to three-fold upregulation of ET receptor A at both mRNA and protein levels in diabetic CCSM. ET-1-induced CCSM contraction is largely dependent upon Rho-kinase (ROK), since it is almost completely blocked by Y-27632 (a highly selective ROK inhibitor). Furthermore, expression of ROKb isoform is selectively upregulated in CCSM from diabetic rabbits. Thus, an increased CCSM tone, modulated by sensitization of the endothelin-mediated contractile pathway via ROK, may be a key component of the molecular mechanism of diabetes-induced erectile dysfunction.

Detrusor smooth muscle (DSM) undergoes hypertrophy after partial bladder outlet obstruction (PBOO) in male rabbits, as it does in men with PBOO induced by benign prostatic hyperplasia. Despite detrusor hypertrophy, some bladders are severely dysfunctional (decompensated). In this study, the rabbit model for PBOO was used to determine the biochemical regulation of the contractile apparatus and force maintenance by the detrusor from decompensated bladders (DB). Bladders from sham-operated rabbits served as a control. On stimulation with 125 mM KCl, the DSM from sham-operated (SB) rabbits showed phasic contractions, whereas the detrusor from DB was tonic, exhibiting slow development of force, a longer duration of force maintenance, and slow relaxation. The Rho kinase (ROK) inhibitor Y-27632 enhanced the relaxation of precontracted DSM strips from DB. The enhancement of relaxation of the KCl-induced contraction of DB by Y-27632 was associated with dephosphorylation of myosin light chain (MLC20). The DSM extract from DB showed low phosphatase activity compared with that from SB. The DB also showed more Ca2+-independent MLC20 phosphorylation, which was partially inhibited by Y-27632. RT-PCR and Western blotting revealed similar expression levels of MLC kinase and ROK-alpha in SB and DB, but ROK-beta was overexpressed in DB. These results suggest that the ROK-mediated pathway is partly responsible for the high degree of force maintenance and slow relaxation in the detrusor from DB.

A tight balance between synaptic vesicle exocytosis and endocytosis is fundamental to maintaining synaptic structure and function. Calcium influx through voltage-gated Ca2+ channels is crucial in regulating synaptic vesicle exocytosis. However, much less is known about how Ca2+ regulates vesicle endocytosis or how the endocytic machinery becomes enriched at the nerve terminal. We report here a direct interaction between voltage-gated Ca2+ channels and endophilin, a key regulator of clathrin-mediated synaptic vesicle endocytosis. Formation of the endophlin-Ca2+ channel complex is Ca2+ dependent. The primary Ca2+ binding domain resides within endophilin and regulates both endophilin-Ca2+ channel and endophilin-dynamin complexes. Introduction into hippocampal neurons of a dominant-negative endophilin construct, which constitutively binds to Ca2+ channels, significantly reduces endocytosis-mediated uptake of FM 4-64 dye without abolishing exocytosis. These results suggest an important role for Ca2+ channels in coordinating synaptic vesicle recycling by directly coupling to both exocytotic and endocytic machineries.

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