Differentiating connexin hemichannels and pannexin channels in cellular ATP release.

Adenosine triphosphate (ATP) plays a fundamental role in cellular communication, with its extracellular accumulation triggering purinergic signaling cascades in a diversity of cell types. While the roles for purinergic signaling in health and disease have been well established, identification and differentiation of the specific mechanisms controlling cellular ATP release is less well understood. Multiple mechanisms have been proposed to regulate ATP release with connexin (Cx) hemichannels and pannexin (Panx) channels receiving major focus. However, segregating the specific roles of Panxs and Cxs in ATP release in a plethora of physiological and pathological contexts has remained enigmatic. This multifaceted problem has arisen from the selectivity of pharmacological inhibitors for Panxs and Cxs, methodological differences in assessing Panx and Cx function and the potential compensation by other isoforms in gene silencing and genetic knockout models. Consequently, there remains a void in the current understanding of specific contributions of Panxs and Cxs in releasing ATP during homeostasis and disease. Differentiating the distinct signaling pathways that regulate these two channels will advance our current knowledge of cellular communication and aid in the development of novel rationally-designed drugs for modulation of Panx and Cx activity, respectively.

Organ of Corti culture for functional analysis of precursor, support and hair cells

Em mamíferos a perda das células ciliadas determina perda auditiva neurosensorial permanente, já que estas células encontram-se em diferenciação terminal e seus precursores não mais entram no ciclo celular...

Loss of hair cells in mammals causes permanent sensorineural hearing loss, as these cells are terminally-differentiated and their precursors do not reenter the cell cycle. The aims of this study were to establish primary cell cultures and subcultures of organ of Corti...

Oleamide derivatives suppress the spontaneous metastasis by inhibiting connexin 26.

We previously reported that overexpressing connexin 26 (Cx26) enhances the spontaneous metastasis of mouse BL6 melanoma cells. In contrast, daily intraperitoneal injections of an oleamide derivative named MI-18 potently inhibits the spontaneous metastasis of BL6 cells. In the present study, we chemically synthesized a novel oleamide derivative named MI-22 and found that it also efficiently suppressed the spontaneous metastasis of BL6 cells. Both MI-18 and MI-22 inhibited the gap junction-mediated intercellular communications (GJIC) that are formed between HeLa cells by the ectopic expression of the hCx26 and hCx32 human connexin subtypes; however, they had no effect on GJIC mediated by hCx40, hCx43 or hCx45. Fluorescently labeled MI-18 primarily localized not only at plasma membrane but also at Golgi/endosome. This suggests that this oleamide derivative may also act on the Cx26 molecules that accumulate in the Golgi/endosome because of their overexpression. Notably, neither derivative had a cytotoxic effect on HeLa cells when they were added into the tissue culture medium. Taken together, we propose that the MI-18 and MI-22 oleamide derivatives may serve as prototypes for novel and clinically important anticancer drugs.

Role of connexin-based gap junction channels in testis.

Spermatogenesis is a highly controlled process that allows proliferation and differentiation of male germ cells. This is under classical endocrine and paracrine controls. There is also evidence that gap junctions between Leydig cells, between Sertoli cells and between Sertoli and germ cells participate in the local regulation of spermatogenesis. Recent studies reveal that connexin 43 (Cx43), the predominant gap junction protein in the testis, is essential for the initiation and maintenance of spermatogenesis. In this review, we focus on the identification, distribution and control of connexins in the mammalian testis. The implication of connexin-based gap junctions in testicular physiology and in pathological disorders of spermatogenesis (spermatogenic arrest and testis cancer) is also discussed.

Connexin26 is responsible for anionic molecule permeability in the cochlea for intercellular signalling and metabolic communications.

Abstract A gap junction is composed of two hemichannels and possesses a relatively large pore size ( approximately 10-15 A), allowing passage of ions and molecules up to 1 kDa. Here, we report that connexin hemichannels and gap junctions in the guinea pig cochlea had significant charge selectivity among permeating molecules. In coincubation with anionic and cationic fluorescent dyes, hemichannel permeability in isolated cochlear supporting cells showed significant charge selectivity; 31% of cells had only cationic dye influx and 6% of cells had only anionic dye influx. Charge-selective influx contrary to dye size was also found, indicating charge as a dominant determinant in permeability. The cell-cell gap junctional permeability was consistent with hemichannel permeability and also showed strong charge selectivity; the permeation of anionic dyes was slower than that of cationic probes in the cochlear sensory epithelium. With a combination of immunofluorescent staining for connexin26 (Cx26) and Cx30, which are the predominant connexin isoforms in the cochlea, Cx26 was demonstrated to correlate with anionic permeability. The data indicated that cochlear gap junctions have strong charge selectivity in molecular permeability and metabolic communication. Cx26 mutation may induce specific, irreparable impairment in intercellular signalling and energy and nutrient supplies in the cochlea, causing cell degeneration and hearing loss, given that many important cell-signalling and nutrient and energy molecules (e.g. IP3, ATP, cAMP and cGMP) are anions.

The detection of hamster connexins: a comparison of expression profiles with wild-type mouse and the cancer-prone Min mouse.

The open reading frames of 17 connexins from Syrian hamster (using tissues) and 16 connexins from the Chinese hamster cell line V79, were fully (Cx30, Cx31, Cx37, Cx43 and Cx45) or partially sequenced. We have also detected, and partially sequenced, seven rat connexins that previously were unavailable. The expression of connexin genes was examined in some hamster organs and cultured hamster cells, and compared with wild-type mouse and the cancer-prone Min mouse. Although the expression patterns were similar for most organs and connexins in hamster and mouse, there were also some prominent differences (Cx29 and 30.3 in testis; Cx31.1 and 32 in eye; Cx46 in brain, kidney and testis; Cx47 in kidney). This suggests that some connexins have species-specific expression profiles. In contrast, there were minimal differences in expression profiles between wild type and Min mice. Species-specific expression profiles should be considered in attempts to make animal models of human connexin-associated diseases.

A simple RT-PCR-based strategy for screening connexin identity

Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx) proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s) can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain) that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins