The analgesic properties of the opium poppy Papever somniferum were first mentioned by Hippocrates around 400 BC, and opioid analgesics remain the mainstay of pain management today. These drugs can cause the serious side-effect of respiratory depression that can be lethal with overdose, however the critical brain sites and neurochemical identity of the neurons mediating this depression are unknown. By locally manipulating neurotransmission in the adult rat, we identify the critical site of the medulla, the preBötzinger complex, that mediates opioid-induced respiratory depression in vivo. Here we show that opioids at the preBötzinger complex cause respiratory depression or fatal apnea, with anesthesia and deep-sleep being particularly vulnerable states for opioid-induced respiratory depression. Importantly, we establish that the preBötzinger complex is fully responsible for respiratory rate suppression following systemic administration of opioid analgesics. The site in the medulla most sensitive to opioids corresponds to a region expressing neurokinin-1 receptors, and we show in rhythmically active brainstem section in vitro that neurokinin-1 receptor-expressing preBötz-inger complex neurons are selectively inhibited by opioids. In summary, neurokinin-1 receptor-expressing preBötzinger complex neurons constitute the critical site mediating opioid-induced respiratory rate depression, and the key therapeutic target for its prevention or reversal.

Background: Intermediate-conductance, calcium-activated potassium channels (IKs) modulate proliferation and differentiation in mesodermal cells by enhancing calcium influx, and they contribute to the physiology of fluid movement in certain epithelia. Previous reports suggest that IK channels stimulate proliferative growth in a keratinocyte cell line; however, because these channels indirectly promote calcium influx, a critically unique component of the keratinocyte differentiation program, an alternative hypothesis is that they would be antiproliferative and pro-differentiating. This study addresses these hypotheses.

Patch-clamp experiments were conducted to study the effects of basal lamina (basement membrane) of preovulatory chicken ovarian follicle on membrane currents in differentiated chicken granulosa cells in a homologous system. The membrane capacitance (measure of total membrane area) was smaller in cells cultured on intact basal lamina than that of control cells. The granulosa cells expressed outward and two inward currents. A small fraction of the cells (3%) expressed only a transient fast-activating and -inactivating inward current carried by Ca(2+). The majority of the cells, however, expressed a slowly activating and inactivating inward current (carried by Cl(-)) that was superimposed on the transient Ca(2+) current. All cells expressed an outward current characteristic of the delayed-rectifier K(+) current. The removal of extracellular Ca(2+) led to elimination of the slow inward Cl(-) current, indicating that it is a Ca(2+)-dependent Cl(-) current. Both peak amplitude and current density of the inward Cl(-) current were significantly lower in cells cultured on freshly isolated intact basal lamina (or basal lamina stored at 4 degrees C for 12 mo) than those of control cells; however, basal lamina had no significant effect on the density of the outward current. Similar to the observations made for intact basal lamina, solubilized basal lamina suppressed the inward Cl(-) current in differentiated granulosa cells. These data show that homologous basal lamina modulates a Ca(2+)-dependent Cl(-) current in differentiated granulosa cells. These findings provide a partial explanation for the mechanisms that subserve the reported effects of basal lamina (basement membrane) on the metabolic functions of differentiated granulosa cells.

Patch clamp experiments were conducted to study the effects of basal lamina (basement membrane) of chicken ovarian follicle on membrane Ca(2+) currents in differentiated chicken granulosa cells in a homologous system. The whole cell patch clamp technique was used to simultaneously monitor membrane capacitance (an indirect measure of total cell surface area) and currents flowing through voltage-dependent Ca(2+) channels (using Ba(2+) as the charge carrier). Membrane capacitance was smaller in cells incubated on intact basal lamina than in control cells (incubated on tissue culture-treated plastic substratum). Granulosa cells expressed both T- and L-type Ca(2+) currents, and the amplitudes of the currents in cells incubated on intact basal lamina were significantly lower than those of control cells. Also, granulosa cells incubated on intact basal lamina were found to have significantly lower T- or L-type Ca(2+) current densities than control cells. Intact basal lamina that had been stored for 12 mo produced effects on T- and L-type Ca(2+) currents similar to those caused by freshly isolated basal lamina. The basal lamina was solubilized completely in one step and used to coat glass coverslips (uncoated glass coverslips served as controls). Granulosa cells incubated on coverslips precoated with solubilized basal lamina assumed spherical shape similar to those incubated on intact basal lamina. Similar to the observations made for intact basal lamina, the solubilized basal lamina suppressed T- and L-type Ca(2+) currents in the differentiated granulosa cells. Moreover, fibronectin, laminin, and type IV collagen, obtained from commercial sources, attenuated T- and L-type Ca(2+) currents in the differentiated granulosa cells. This interplay between basal lamina and Ca(2+) currents may be one mechanism that subserves the effects of the matrix material on metabolic functions of granulosa cells.

The effects of low Ca(2+) on ion currents in hen ovarian granulosa cells were examined. A fast activating and inactivating transient outward current (TOC) and a slowly activating outward current (SOC) could be observed. In the presence of normal Ca(2+) concentration (2. 5 mM) and with a holding potential of -80 mV, SOC was activated in all cells with command pulses more positive than -20 mV. In 2.5 mM Ca(2+), TOC appeared in 10% of cells at the command pulse of +80 mV and in 60-85% of cells at +100 to +120 mV. In low-Ca(2+) solution and command potential of +80 mV (holding potential of -80 mV), the amplitude of TOC was enhanced in cells that expressed it in normal Ca(2+), and TOC appeared in 43% of the cells that did not express it initially in normal Ca(2+). At both normal and low Ca(2+) levels, TOC decreased as the holding potential became more positive. TOC was reduced in Cl(-)-deficient solution and in the presence of 5-nitro-2-(3-phenylpropylamino)benzoic acid, a Cl(-) channel blocker. These findings suggest that chicken granulosa cells express a Ca(2+)-inactivated TOC carried by Cl(-). This current may serve as a signal for some of the reduced metabolic functions of granulosa cells associated with Ca(2+) deficiency.