Natural occurrence of myofiber cytoplasmic enlargement accompanied by decrease in myonuclear number.

It has been shown that changes in the nuclear number in myofibers are synchronized with myofiber size. Therefore, under some conditions, the myonuclear number is thought to be a determinant factor of myofiber size. However, we have clearly shown that denervation-induced fiber atrophy occurs without any decrease in myonuclear number, indicating that the myonuclear number is not always an important determinant factor of myofiber size. However, this was an event found under experimental conditions. In the present study, we examined the morphological features of single myofibers under normal conditions throughout the lifespan of normal mice. We discovered that the C/N ratio (cell volume/nucleus) greatly increases during the growth period and clearly decreases during the aging period. From 5 weeks to 6 months old, the myofibers undergo fiber hypertrophy accompanied by a decrease in myonuclear number. In muscle at 18 months, we found no correlation between myonuclear number and fiber cross-sectional area. These results suggest that, even under normal physiological conditions, the myonuclear number is not always a determinant factor of the myofiber size.

No decrease in myonuclear number after long-term denervation in mature mice.

Age-related but not artificially induced muscle fiber atrophy has been shown to occur without any decrease in myonuclear number, although these results remain controversial. The present study was carried out to clarify whether age difference affects the degree of decrease in myonuclear number occurring with denervation-induced fiber atrophy. After denervation of 3-wk-old (young) and 4-mo-old (mature) mice, single myofibers were isolated from the plantaris muscles by alkali maceration, and their fiber cross-sectional area (CSA), myonuclear number, and cytoplasm-to-myonucleus (C/N) ratios were analyzed. Fiber CSA in both young and mature mice decreased with denervation. Myonuclear number decreased in young mice 5 and 10 days after denervation but was unchanged in mature mice 10 and 120 days after denervation. C/N ratio decreased in mature mice but was unchanged in denervated young mice. These results suggest that age differences affect the degree of decrease of myonuclear number with denervation and that fiber cytoplasmic atrophy may occur without decrease in myonuclear number.

Pharmacologic characterization of the oxytocin receptor in human uterine smooth muscle cells.

[(3)H]-oxytocin was used to characterize the oxytocin receptor found in human uterine smooth muscle cells (USMC). Specific binding of [(3)H]-oxytocin to USMC plasma membranes was dependent upon time, temperature and membrane protein concentration. Scatchard plot analysis of equilibrium binding data revealed the existence of a single class of high-affinity binding sites with an apparent equilibrium dissociation constant (K(d)) of 0.76 nM and a maximum receptor density (B(max)) of 153 fmol mg(-1) protein. The Hill coefficient (n(H)) did not differ significantly from unity, suggesting binding to homogenous, non-interacting receptor populations. Competitive inhibition of [(3)H]-oxytocin binding showed that oxytocin and vasopressin (AVP) receptor agonists and antagonists displaced [(3)H]-oxytocin in a concentration-dependent manner. The order of potencies for peptide agonists and antagonists was: oxytocin>[Asu(1,6)]-oxytocin>AVP= atosiban>d(CH(2))(5)Tyr(Me)AVP>[Thr(4),Gly(7)]-oxytocin>dDAVP, and for nonpeptide antagonists was: L-371257>YM087>SR 49059>OPC-21268>SR 121463A>OPC-31260. Oxytocin significantly induced concentration-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and hyperplasia in USMC. The oxytocin receptor antagonists, atosiban and L-371257, potently and concentration-dependently inhibited oxytocin-induced [Ca(2+)](i) increase and hyperplasia. In contrast, the V(1A) receptor selective antagonist, SR 49059, and the V(2) receptor selective antagonist, SR 121463A, did not potently inhibit oxytocin-induced [Ca(2+)](i) increase and hyperplasia. The potency order of antagonists in inhibiting oxytocin-induced [Ca(2+)](i) increase and hyperplasia was similar to that observed in radioligand binding assays. In conclusion, these data provide evidence that the high-affinity [(3)H]-oxytocin binding site found in human USMC is a functional oxytocin receptor coupled to [Ca(2+)](i) increase and cell growth. Thus human USMC may prove to be a valuable tool in further investigation of the physiologic and pathophysiologic roles of oxytocin in the uterus. British Journal of Pharmacology (2000) 129, 131 - 139

Pharmacological profile of YM087, a novel potent nonpeptide vasopressin V1A and V2 receptor antagonist, in vitro and in vivo.

The biochemical and pharmacological profile of YM087, 4'-[(2-methyl-1,4,5,6-tetrahydroimidazo[4,5-d][1]benzazepin- 6-yl)-carbonyl]-2-phenylbenzanilide monohydrochloride, a newly synthesized nonpeptide vasopressin (AVP) antagonist, was investigated in several in vitro and in vivo studies. YM087 showed high affinity for V1A receptors from rat liver and V2 receptors from rat kidney with Ki values of 0.48 and 3.04 nM, respectively. YM087 also inhibited [3H]oxytocin (OT) binding to rat uterus (OT receptors) plasma membranes with a Ki value of 44.4 nM, and at 100 microM did not affect the binding of [3H]AVP to anterior pituitary (V1B receptors) plasma membranes, which indicated that it had less affinity for these OT and V1B receptors. YM087 had no effect on cytosolic free calcium concentration ([Ca++]i) itself, but suppressed AVP-induced increase in [Ca++]i of cultured vascular smooth muscle cells at the same concentrations as the binding affinities. Furthermore, YM087 potently blocked AVP-induced cAMP production of cultured renal epithelium cells concentration dependently and had no agonistic activities. In in vivo studies, intravenous administration of YM087 inhibited the pressor response to exogenous AVP in pithed rats and produced an aquaretic effect in dehydrated conscious rats in a dose-dependent manner. These results demonstrate that YM087 is a potent and nonpeptide dual AVP V1A and V2 receptors antagonist and can be used in future studies to help clarify the physiological and pathophysiological roles of AVP.