Activation of the complement system on the cell surface results in the insertion of pore forming membrane attack complexes (MAC, C5b‐9). In order to protect themselves from the complement attack, the cells express several regulatory molecules, including the terminal complex regulator CD59 that inhibits assembly of the large MACs by inhibiting the insertion of additional C9 molecules into the C5b‐9 complex. Using the whole cell patch clamp method, we were able to measure accumulation of homologous MACs in the membrane of CD59− human B‐cells, which formed non‐selective ion channels with a total conductance of 360 ± 24 pS as measured at the beginning of the steady‐state phase of the inward currents. C5b‐8 and small‐size MAC (MAC containing only a single C9) can also form ion channels. Nevertheless, in CD59+ human B‐cells in spite of small‐size MAC formation, an ion current could not be detected. In addition, restoring CD59 to the membrane of the CD59− cells inhibited the serum‐evoked inward current. The ion channels formed by the small‐size MAC were therefore sealed, indicating that CD59 directly interfered with the pore formation of C5b‐8 as well as that of small‐size C5b‐9. These results offer an explanation as to why CD59‐expressing cells are not leaky in spite of a buildup of homologous C5b‐8 and small‐size MAC. Our experiments also confirmed that ion channel inhibition by CD59 is subject to homologous restriction and that CD59 cannot block the conductivity of MAC when generated by xenogenic (rabbit) serum.

Activation of neurons in the midbrain periaqueductal gray (PAG) inhibits spinal dorsal horn neurons and produces behavioral antinociception in animals and analgesia in humans. Although dorsal horn regions modulated by PAG activation contain all three opioid receptor classes (mu, delta, and kappa), as well as enkephalinergic interneurons and terminal fields, descending opioid-mediated inhibition of dorsal horn neurons has not been demonstrated. We examined the contribution of dorsal horn mu-opioid receptors to the PAG-elicited descending modulation of nociceptive transmission. Single-unit extracellular recordings were made from rat sacral dorsal horn neurons activated by noxious heating of the tail. Microinjections of bicuculline (BIC) in the ventrolateral PAG led to a 60-80% decrease in the neuronal responses to heat. At the same time, the responses of the same neurons to iontophoretically applied NMDA or kainic acid were not consistently inhibited. The inhibition of heat-evoked responses by PAG BIC was reversed by iontophoretic application of the selective mu-opioid receptor antagonists, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP). A similar effect was produced by naloxone; however, naloxone had an excitatory influence on dorsal horn neurons in the absence of PAG-evoked descending inhibition. This is the first demonstration that endogenous opioids acting via spinal mu-opioid receptors contribute to brain stem control of nociceptive spinal dorsal horn neurons. The inhibition appears to result in part from presynaptic inhibition of afferents to dorsal horn neurons.

Modulation of sacral spinal dorsal horn neurons by the ventrolateral PAG was studied by extracellular recording combined with microiontophoretic applications of alpha-adrenergic agonists or antagonists. Bicuculline (BIC, 15 ng) microinjected into the ventrolateral PAG produced a consistent inhibition of the responses of nociceptive dorsal horn neurons. After PAG-BIC applications, the total number of spikes per heat stimulation period was significantly decreased to a mean of 37 +/- 19% (n = 8) of the pre-BIC control. Local iontophoresis of the selective alpha2-adrenoceptor antagonists idazoxan or yohimbine but not the selective alpha1 antagonist benoxathian significantly reversed PAG-BIC-evoked inhibition. At low ejection currents, clonidine, an alpha2-adrenoceptor agonist, markedly reduced noxious heat-evoked responses but had no consistent action on the responses to iontophoresed excitatory amino acids [EAA; N-methyl--aspartate (NMDA) or kainic acid]. At ejection currents higher than required to block descending inhibition, idazoxan potentiated responses to both heat and EAA iontophoresis. At higher ejection currents, EAA responses were inhibited by clonidine. This indicates that both presynaptic and postsynaptic alpha2 receptors are capable of inhibiting the recorded neurons. Activation of the alpha1 adrenoceptors by iontophoresis of methoxamine often led to a marked increase in the responses to kainic acid and, to a lesser extent, to NMDA iontophoresis or noxious heat. Together with previously reported work, the current experiments demonstrate that PAG neurons inhibit nociceptive dorsal horn neurons primarily through an indirect alpha2 adrenoceptor mechanism. In this same population of dorsal horn neurons, norepinephrine has a direct alpha1-mediated excitatory effect.