Effects of elevated cytosolic calcium on ACh-induced swine tracheal smooth muscle contraction

Increased intracellular calcium concentration ([Ca²⁺]_i) is required for smooth muscle contraction. In tracheal and other tonic smooth muscles, contraction and elevated [Ca²⁺]_i are maintained as long as an agonist is present. To evaluate the physiological role of steady-state increases in Ca²⁺ on tension maintenance, [Ca²⁺]_i was elevated using ionomycin, a Ca²⁺ ionophore or charybdotoxin, a large-conductance calcium-activated potassium channel (K_Ca) blocker prior to or during exposure of tracheal smooth muscle strips to Ach (10^–9 to 10^–4 M). Ionomycin (5 µM) in resting muscles induced increases in [Ca²⁺]_i to 500±230 nM and small increases in force of 2.6±2.3 N/cm². This tension is only 10% of the maximal tension induced by ACh. Charybdotoxin had no effect on [Ca²⁺]_i or tension in resting muscle. After pretreatment of muscle with ionomycin, the concentration-response relationship for ACh-induced changes in tension shifted to the left (EC₅₀=0.07±0.05 µM ionomycin; 0.17±0.07 µM, control, p<0.05). When applied to the muscles during steady-state responses to submaximal concentrations of ACh, both ionomycin and charybdotoxin induced further increases in tension. The same magnitude increase in tension occurs after ionomycin and charybdotoxin treatment, even though the increase in [Ca²⁺]_i induced by charybdotoxin is much smaller than that induced by ionomycin. We conclude that the resting muscle is much less sensitive to elevation of [Ca²⁺]_i when compared to muscles stimulated with ACh. Steady-state [Ca²⁺]_i limits tension development induced by submaximal concentrations of ACh. The activity of K_Ca moderates the response of the muscle to ACh at concentrations less than 1 µM.     

Further characterization of nociception-related and arterial pressure-related neuronal responses in the nucleus reticularis gigantocellularis of the rat

The present study was undertaken to further characterize the nucleus reticularis gigantocellularis (NRGC) of the medulla oblongata in the central processing of nociceptive and cardiovascular signals, and its modulation by metenkephalin. In Sprague-Dawley rats anesthetized with pentobarbital sodium, we found that all 125 spontaneously active NRGC neurons that responded to noxious stimuli (tail clamp) also exhibited arterial pressure-relatedness. Forty neurons additionally manifested cardiac periodicity that persisted even during nociceptive responses. While maintaining their cardiovascular responsive characteristics, the nociception-related NRGC neuronal activity was blocked, naloxone-reversibly (0.5 mg/kg, i.v.), by morphine (5 mg/kg, i.v.). Microiontophoretically applied met-enkephalin suppressed the responsiveness of NRGC neurons to individually delivered tail clamp or transient hypertension induced by phenylephrine (5 µg/kg, i.v.). Interestingly, in NRGC neurons that manifested both nociception and arterial pressure relatedness, the preferential reduction in the response to noxious stimuli upon simultaneous elevation in systemic arterial pressure was reversed to one that favored nociception in the presence of met-enkephalin. All actions of met-enkephalin were discernibly blocked by the opioid receptor antagonist, naloxone. Our results suggest that individual NRGC neurons may participate in the processing of both nociceptive and cardiovascular information, or in the coordination of the necessary circulatory supports during nociception. In addition, neuropeptides such as met-enkephalin may exert differential modulation on neuronal responsiveness according to the prevailing physiologic status of the animal. They also showed that NRGC may be a central integrator for pain and cardiovascular-related functions.     

Yeast Kre1p is a cell surface O-glycoprotein

The Saccharomyces cerevisiae KRE1 gene encodes a secretory protein required for the production of the cell wall polymer (1 6)--glucan. Here we report further characterization of the KRE1 gene product, Krelp. A functional, epitope-tagged Krelp is shown to be highly modified in a SEC53-dependent manner. Krelp is O-glycosylated, but the basis for the majority of its post-translational modification is unknown. Fractionation of Kre1p reveals a cell wall-associated form and a less abundant membrane-associated species. Indirect immunoflurorescence demonstrates that Kre1p localizes to the cell surface, where it becomes concentrated at the surface of mother cells. Such a localization of Kre1p seems to parallel the CAL1/CSD2-dependent cell wall deposition of chitin found in S. cerevisiae, and is consistent with evidence from Schizophyllum commune that (1 6)--glucan accumulates during maturation of the subapical region of the wall distal to the hyphal tip.     

In Vitro Reconstitution of Cortical Actin Assembly Sites in Budding Yeast

We have developed a biochemical approach for identifying the components of cortical actin assembly sites in polarized yeast cells, based on a permeabilized cell assay that we established for actin assembly in vitro. Previous analysis indicated that an activity associated with the cell cortex promotes actin polymerization in the bud. After inactivation by a chemical treatment, this activity can be reconstituted back to the permeabilized cells from a cytoplasmic extract. Fractionation of the extract revealed that the reconstitution depends on two sequentially acting protein factors. Bee1, a cortical actin cytoskeletal protein with sequence homology to Wiskott-Aldrich syndrome protein, is required for the first step of the reconstitution. This finding, together with the severe defects in actin organization associated with the bee1 null mutation, indicates that Bee1 protein plays a direct role in controlling actin polymerization at the cell cortex. The factor that acts in the second step of the reconstitution has been identified by conventional chromatography. It is composed of a novel protein, Pca1. Sequence analysis suggests that Pca1 has the potential to interact with SH3 domain-containing proteins and phospholipids.     

Oligosyndactyly: A lethal mutation in the mouse that results in mitotic arrest very early in development

The mutation, oligosyndactyly, results in syndactyly, muscle anomalies, and diabetes insipidus in heterozygous mice. When homozygous, the mutation is lethal early in development. Although homozygous embryos are able to form blastocyst outgrowths (the in vitro equivalent to implantation), cells begin to accumulate in mitosis as early as the blastocyst stage. Even though the cytologic appearance is that of mitotic cells treated with a microtubule inhibitor such as colcemid, the homozygous embryos do, in fact, have normal appearing mitotic spindles. These results define the Os mutation as one which, in the homozygous state, prevents the movement of chromosomes from the metaphase plate. It is the first mammalian developmental mutation to be so defined and is unique among all mitotic arrest mutations thus far described in higher eucaryotes.     

Variation in testosterone production among boards and its relationship to sexual interest and breeding performance

Variation in ability to produce testosterone in response to both GnRH and ACTH administration and quatitative relationships between GnRH-stimulated testosterone levels, ACTH-stimulated testosterone levels, sexual interest and breeding performance were assessed in a group of 31 Duroc boars (115.4 +/- 2.5 kg body weight and 212.2 +/- 3.0 days of age). Mean area beneath the testosterone response curve increased (P<0.01) after GnRH and ACTH but the magnitude of response was variable among boars. Post-GnRH testosterone area varied from 7.44 to 50.86 ng/ml X h with a CV = 52.41% while post-ACTH testosterone area varied from 4.99 to 28.78 ng/ml X h with a CV = 45.46%. Mean sexual interest and mean breeding performance scores were correlated (r = 0.67, P<0.01); however, correlations of either variable with testosterone areas were low and nonsignificant. These results indicate that the testosterone-producing ability of boars of similar age and breeding is highly variable and suggest that peripheral testosterone concentrations may not be good indicators of either libido or breeding performance.