Innervation of the anococcygeus muscle of the rat

Summary The innervation of the anococcygeus muscle of the rat was investigated with regard to the histochemical features of nerve fibers within the muscle and to the location of the postganglionic autonomic neurons which are the source of these fibers. Acetylcholinesterase-positive fibers and catecholaminergic fibers are abundant in the anococcygeus as well as the related retractor penis muscle. Neuronal somata, either between muscle bundles of the anococcygeus or in the connective tissue sheath, are also acetylcholinesterase-positive. Nerve fibers and a minority of the ganglion cells in the anococcygeus and retractor penis muscles are immunoreactive for vasoactive intestinal polypeptide. Injection of the retrogradely transported dye Fluorogold into the anococcygeus muscle filled neurons in the abdominopelvic sympathetic chain, pelvic plexus and a small number of neurons in the inferior mesenteric ganglion. In the pelvic plexus, some neurons were located in the major pelvic ganglion but most were found along the main penile nerve and its branches to the anococcygeus muscle. Immunocytochemistry of these identified neurons indicates that about one half of them are positive for vasoactive intestinal polypeptice. These results raise the possibility that both acetylcholine and vasoactive intestinal polypeptide are important neurotransmitters in autonomic nerves to the anococcygeus muscle.     

On the ultrastructure of the rat anococcygeus muscle

Cell and Tissue Research - The rat anococcygeus muscle, which is known from previous functional and histochemical investigations to be a smooth muscle with a dense adrenergic innervation, was...     

The effect of palytoxin on neuromuscular junctions in the anococcygeus muscle of the rat

Palytoxin, a highly toxic natural product isolated from zoanthids of the genus Palythoa, is accumulated by a wide range of fishes and marine invertebrates used as food in the Indo-Pacific. It is responsible for many incidents of human morbidity and mortality. The toxin is a potent smooth muscle spasmogen. The cause of the contraction of smooth muscle is unclear, but recent work strongly suggests that it is primarily initiated by the release of neurotransmitters from the motor innervation of the smooth muscle. We show here that palytoxin caused the swelling of the muscle cells and some internal organelles of the anococcygeus muscle of the rat, but no substantial structural damage to the tissue. Axons and Schwann cells were also swollen but the most dramatic feature was the depletion of synaptic vesicles from putative release sites in the axons. Some axons were physically damaged following exposure to the toxin, but this was relatively uncommon (<10% of all axons studied). In the majority of axons there was no damage to nerve terminal membranes, but there was damage to mitochondria. The depletion of vesicles involved all types – clear, dense-cored, large and small. Our observations and pharmacological data gathered elsewhere, provide a neuropathological basis for the spasmogenic activity of palytoxin.     

Ultrastructural identification of non-adrenergic,non-cholinergic nerves in the rat anococcygeus muscle

The innervation of the rat anococcygeus muscle has been investigated ultrastructurally following fixation with a modified chromaffin reaction for the demonstration of biogenic amines (Tranzer and Richards, 1976). Three types of nerve profiles were revealed: (1) 60-70% of the profiles are adrenergic; (2) less than 5% of the profiles appear to be cholinergic; (3) up to 40% of the profiles are distinguished by the presence of a characteristically high proportion of electron-opaque, chromaffin-negative vesicles, 85-110 nm in diameter. This third type of profile was not affected by 6-OHDA, and is considered to represent the non-adrenergic, non-cholinergic inhibitory innervation of this tissue. Because of the morphological similarity of this nerve type, apart from the smaller vesicle size, to classical peptidergic nerve endings, they have been termed "small p-type" (sp-type). These results are discussed in relation to a previous report describing only two types of nerve profiles in this tissue (Gillespie and Lüllmann-Rauch, 1974).     

Peroxynitrite inhibits relaxation and induces pulmonary artery muscle contraction in the newborn rat

Nitric oxide (NO) reacts with superoxide anion to form the peroxynitrite anion (ONOO-), a molecule with pulmonary vasodilator properties in the adult rat. The purpose of this study was to compare the effects of ONOO- on intrapulmonary arteries from the newborn (days 4-7), juvenile (day 14), and adult rat. Following thromboxane A2 (TXA2) analogue (U46619) prestimulation, newborn vessels were more sensitive to ONOO- -induced muscle contraction, compared to both the juvenile and the adult vessels. Peroxynitrite-induced contraction in newborn vessels was abrogated by ibuprofen, an endothelin B-receptor blocker (A-192621), or a rho-kinase-specific inhibitor (Y27632) (all p < 0.01). Following KCl stimulation and TXA2 receptor blockade, ONOO- induced NO-dependent muscle relaxation in newborn vessels via stimulation of the endothelial and inducible nitric oxide synthases. However, in the presence of ONOO-, the pulmonary artery relaxation response to endothelium-dependent stimulation was significantly reduced (p < 0.01). Finally, newborn but not adult pulmonary arteries exposed to ONOO- showed a 10-fold increase in 8-isoprostane production, a possible mediator of ONOO- -induced contraction. We conclude that exposure to ONOO- results in a unique response in newborn intrapulmonary arteries characterized by increased 8-isoprostane generation, which we believe is responsible for its vasoconstrictor effect. This unique response potentially renders the newborn more susceptible to ONOO- -induced pulmonary hypertension than older animals.     

Effects of TRIM on tension, intracellular calcium and nitrergic transmission in the rat anococcygeus muscle.

The effects of the putatively selective inhibitor of neuronal nitric oxide synthase (nNOS) 1-(2-trifluoromethylphenyl) imidazole (TRIM) were investigated on contractility, intracellular calcium and nitrergic relaxations in the rat anococcygeus muscle. TRIM (100-1000 microM) reduced the tension of rat anococcygeus muscles when contracted with guanethidine (10 microM) and clonidine (0.1 microM). Relaxations to TRIM persisted in the presence of the non-selective NOS inhibitor L-NAME (100 microM) and the inhibitor of soluble guanylate cyclase ODQ (1 microM). TRIM also reduced tension when muscles were contracted with phenylephrine (3 microM), noradrenaline (3 microM) or high K physiological salt solution (high KPSS; 60mM). Influx of calcium ([Ca(2+)](i)) in response to high KPSS was significantly reduced in the presence of TRIM (1mM). TRIM also inhibited the influx of (45)Ca(2+) induced by KPSS, but had no effect on the influx induced by phenylephrine (10 microM). TRIM (300 microM) had a modest, but significant, inhibitory effect on nitrergic relaxations that were evoked by electrical field stimulation (1-10 Hz, 15 V, 10s trains) in muscles contracted with guanethidine and clonidine. In contrast, L-NAME (1-100 microM) inhibited these nitrergic responses with an IC(50) of 9.31+/-0.87 microM (n=4). The results suggest that the smooth muscle relaxant effect of TRIM in the rat anococcygeus muscle may affect the entry of Ca(2+) possibly through voltage-operated calcium channels. Furthermore, the relatively modest effect of TRIM on nitrergic responses indicates that it is not a particularly reliable inhibitor of nNOS.     

Occurrence of uranaffin-positive synaptic vesicles in both adrenergic and non-adrenergic nerves of the rat anococcygeus muscle

Summary The uranaffin reaction in rat anococcygeus muscle, which receives a dual innervation of both adrenergic and non-cholinergic, non-adrenergic nerves was examined. Dense reaction product was observed in the vesicular membranes and/or the cores of some synaptic vesicles in the adrenergic nerve terminals. Occasional vesicles were filled up with dense reaction product. In the prominent population of small clear vesicles, however, no dense reaction product was observed. The number of small granular vesicles in the adrenergic nerve terminals was markedly increased after the administration of 5-hydroxydopamine (5-OHDA). These granular vesicles were moderately stained with uranaffin deposit on the cores but their limiting membranes possessed no uranaffin deposit at all.In the non-adrenergic nerve terminals, on the other hand, uranaffin deposit of variable density was observed on the cores of large granular vesicles but never on their limiting membranes or on the small clear vesicles. There was no change in the axon profiles after the administration of 5-OHDA.The possible occurrence of purines in the cores of large granular vesicles in the non-adrenergic nerves is discussed.     

Peroxynitrite induces apoptosis in rat aortic smooth muscle cells: possible relation to vascular diseases

An emerging body of evidence is accumulating to suggest that in vivo formation of free radicals in the vasculature, such as peroxynitrite (ONOO-), and programmed cell death (i.e., apoptosis) play important roles in vascular diseases such as atherosclerosis, hypertension, and restenosis. The present study was designed to determine whether primary rat aortic smooth muscle cells (SMCs) undergo apoptosis following treatment with ONOO-. Direct exposure of primary rat aortic SMCs to ONOO--induced apoptosis in a concentration-dependent manner, as confirmed by means of quantitative fluorescence staining and TUNEL assays. ONOO--induced apoptosis in rat aortic SMCs appears to involve activation of Ca2+-dependent endonucleases. Although the precise mechanisms by which peroxynitrite induces apoptosis in rat aortic SMCs need to be further investigated, the present, preliminary findings could be used to suggest that ONOO- formation in the vasculature may play roles in the processes of vascular diseases, such as atherosclerosis, hypertension, and restenosis, via adverse actions on blood vessels.     

Long-term isoprenaline administration produces an increase in capillarity in the soleus muscle of the rat

The effects of isoprenaline administration (300 micrograms/kg for 5 weeks) on rat soleus muscle capillarity and glycolytic and oxidative capacities were evaluated. The treatment resulted in ventricular hypertrophy. The activities of lactic dehydrogenase, pyruvate kinase, citrate synthase, and cytochrome c oxidase in soleus muscle homogenates were not different between control and isoprenaline-injected animals. Capillaries were visualized in muscle cross sections treated to demonstrate ATPase activity after acid preincubation. Capillary density was higher in the experimental (873 +/- 38 capillaries/mm2) than in the control (713 +/- 33 capillaries/mm2) animals. Capillary to fiber ratio was also higher in the experimental (2.47 +/- 0.10) than in control (2.09 +/- 0.08) animals, but fiber cross-sectional area was not changed by the treatment (2836 +/- 87 microns2 in controls and 2951 +/- 136 microns2 in experimental). A plot of capillary to fiber ratio vs. fiber cross-sectional area showed that at a given fiber cross-sectional area the value of capillary to fiber ratio of the treated animals was higher than that of the controls. This indicates that treatment resulted in the proliferation of microvessels. The results suggest that prolonged beta-adrenergic stimulation results in the development of new capillaries but that this is not accompanied by increases in the oxidative capacity of the soleus muscle of the rat.     

Peroxynitrite has potent pulmonary vasodilator activity in the rat

Peroxynitrite (PN) worsens pathological conditions associated with oxidative stress. However, beneficial effects have also been reported. PN has been shown to demonstrate vasodilator as well as vasoconstrictor properties that are dependent upon the experimental conditions and the vascular bed studied. PN-induced vascular smooth muscle relaxation may involve the formation of nitric oxide (NO) donors. The present results show that PN has significant vasodilator activity in the pulmonary and systemic vascular beds, and that responses to PN were not attenuated by L-penicillamine (L-PEN), a PN scavenger, whereas responses to sodium nitroprusside (SNP) were decreased. PN had a small inhibitory effect on decreases in arterial pressure in response to the NO donors diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) and S-nitrosoglutathione (GSNO). PN partially reversed hypoxic pulmonary vasoconstriction. PN responses were attenuated by the soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and responses to PN and the PN precursor, 3-morpholinosydnonimine (SIN-1), were different. These data show that PN has potent pulmonary vasodilator activity in the rat, and provide evidence that a PN interaction with S-nitrosothiols is not the major mechanism mediating the response. These data suggest that responses to PN are mediated by the activation of sGC, and that PN has a small inhibitory effect on NO responses.     

Peroxynitrite irreversibly decreases diastolic and systolic function in cardiac muscle

Much of the damaging action of nitric oxide in heart may be due to its diffusion-limited reaction with superoxide to form peroxynitrite. Direct infusion of peroxynitrite into isolated perfused hearts fails to model the effects of in situ formation because the bulk of peroxynitrite decomposes before reaching the myocytes. To examine the direct effects of peroxynitrite on the contractile apparatus of the heart, we exposed intact and skinned rat papillary muscles to a steady state concentration of 4-microM peroxynitrite for 5 min, followed by a 30-min recovery period to monitor irreversible effects. In intact muscles developed force fell immediately to 26% of initial force, recovering to 43% by 30 min. Resting tension increased by 600% immediately, and was still elevated 500% by 30 min. Nitrotyrosine immunochemistry showed that peroxynitrite can induce tyrosine nitration at low concentrations and is capable of penetrating 200-380 microm into the papillary muscle after a 5-min infusion. Decomposed peroxynitrite had no effect on either intact or skinned muscle developed force or resting tension. Our results show that peroxynitrite directly damages both developed force and resting tension of isolated heart muscle, which can be extrapolated to systolic and diastolic injury in intact hearts.     

Peroxynitrite reversibly inhibits Ca(2+)-activated K(+) channels in rat cerebral artery smooth muscle cells

Peroxynitrite (ONOO(-)) is a contractile agonist of rat middle cerebral arteries. To determine the mechanism responsible for this component of ONOO(-) bioactivity, the present study examined the effect of ONOO(-) on ionic current and channel activity in rat cerebral arteries. Whole cell recordings of voltage-clamped cells were made under conditions designed to optimize K(+) current. The effects of iberiotoxin, a selective inhibitor of large-conductance Ca(2+)-activated K(+) (BK) channels, and ONOO(-) (10-100 microM) were determined. At a pipette potential of +50 mV, ONOO(-) inhibited 39% of iberiotoxin-sensitive current. ONOO(-) was selective for iberiotoxin-sensitive current, whereas decomposed ONOO(-) had no effect. In excised, inside-out membrane patches, channel activity was recorded using symmetrical K(+) solutions. Unitary currents were sensitive to increases in internal Ca(2+) concentration, consistent with activity due to BK channels. Internal ONOO(-) dose dependently inhibited channel activity by decreasing open probability and mean open times. The inhibitory effect of ONOO(-) could be overcome by reduced glutathione. Glutathione, added after ONOO(-), restored whole cell current amplitude to control levels and reverted single-channel gating to control behavior. The inhibitory effect of ONOO(-) on membrane K(+) current is consistent with its contractile effects in isolated cerebral arteries and single myocytes. Taken together, our data suggest that ONOO(-) has the potential to alter cerebral vascular tone by inhibiting BK channel activity.     

A comparison of the effects of morphine withdrawal, thyroxine or thyroidectomy on the sensitivity of the anococcygeus muscle to agonists and on serum thyroxine levels in the rat

The effects of morphine withdrawal, thyroxine or thyroidectomy on the plasma levels of thyroxine and on the sensitivity of the anococcygeus muscle to acetylcholine and noradrenaline were investigated in the rat. Morphine withdrawal or thyroidectomy increased the maximum response of both agonists and morphine withdrawal increased the pD₂ value for acetylcholine. Thyroxine did not affect the maximum responses but increased the pD₂ values for both agonists. Plasma thyroxine levels were raised by thyroxine administration and were reduced by thyroidectomy and also during morphine administration and withdrawal.     

Effect of streptozotocin diabetes on motor and inhibitory transmission in rat anococcygeus.

The effect of streptozotocin diabetes of 4-week duration on the adrenergic motor transmission and on the nonadrenergic, noncholinergic, inhibitory transmission in the rat anococcygeus was investigated by recording contractile and relaxant activity of isolated muscle preparations taken from diabetic and age-matched control animals. The neurogenic contractile responses to electrical field stimulation were significantly reduced in the preparations from diabetic rats. The inhibitory transmission remained unaffected in the diabetic rats. Concentration--response curves showed no change in sensitivity of the diabetic anococcygei to noradrenaline. The maximum tension generated was also similar in preparations from diabetic and nondiabetic animals. The contractile responses to electrical field stimulation were significantly greater in preparations from diabetic rats treated for 4 weeks with either sorbinil (20 mg.kg-1.day-1 orally) or myo-inositol (667 mg.kg-1.day-1 orally) when compared with the untreated diabetic controls; the sensitivity to noradrenaline was identical in all three groups. It is concluded that streptozotocin diabetes causes a significant reduction of adrenergic contractile responses of the anococcygeus to electrical field stimulation by a prejunctional mechanism, and the reduction can be prevented by treating the animals with the aldose reductase inhibitor sorbinil or with myo-inositol.     

Fast and slow gating relaxations in the muscle chloride channel CLC-1

Gating of the muscle chloride channel CLC-1 involves at least two processes evidenced by double-exponential current relaxations when stepping the voltage to negative values. However, there is little information about the gating of CLC-1 at positive voltages. Here, we analyzed macroscopic gating of CLC-1 over a large voltage range (from -160 to +200 mV). Activation was fast at positive voltages but could be easily followed using envelope protocols that employed a tail pulse to -140 mV after stepping the voltage to a certain test potential for increasing durations. Activation was biexponential, demonstrating the presence of two gating processes. Both time constants became exponentially faster at positive voltages. A similar voltage dependence was also seen for the fast gate time constant of CLC-0. The voltage dependence of the time constant of the fast process of CLC-1, tau(f), was steeper than that of the slow one, tau(s) (apparent activation valences were z(f) approximately -0. 79 and z(s) approximately -0.42) such that at +200 mV the two processes became kinetically distinct by almost two orders of magnitude (tau(f) approximately 16 micros, tau(s) approximately 1 ms). This voltage dependence is inconsistent with a previously published gating model for CLC-1 (Fahlke, C., A. Rosenbohm, N. Mitrovic, A.L. George, and R. Rüdel. 1996. Biophys. J. 71:695-706). The kinetic difference at 200 mV allowed us to separate the steady state open probabilities of the two processes assuming that they reflect two parallel (not necessarily independent) gates that have to be open simultaneously to allow ion conduction. Both open probabilities could be described by Boltzmann functions with gating valences around one and with nonzero "offsets" at negative voltages, indicating that the two "gates" never close completely. For comparison with single channel data and to correlate the two gating processes with the two gates of CLC-0, we characterized their voltage, pH(int), and [Cl](ext) dependence, and the dominant myotonia inducing mutation, I290M. Assuming a double-barreled structure of CLC-1, our results are consistent with the identification of the fast and slow gating processes with the single-pore and the common-pore gate, respectively.