Gap junctions and cochlear homeostasis.

Gap junctions play a critical role in hearing and mutations in connexin genes cause a high incidence of human deafness. Pathogenesis mainly occurs in the cochlea, where gap junctions form extensive networks between non-sensory cells that can be divided into two independent gap junction systems, the epithelial cell gap junction system and the connective tissue cell gap junction system. At least four different connexins have been reported to be present in the mammalian inner ear, and gap junctions are thought to provide a route for recycling potassium ions that pass through the sensory cells during the mechanosensory transduction process back to the endolymph. Here we review the cochlear gap junction networks and their hypothesized role in potassium ion recycling mechanism, pharmacological and physiological gating of cochlear connexins, animal models harboring connexin mutations and functional studies of mutant channels that cause human deafness. These studies elucidate gap junction functions in the cochlea and also provide insight for understanding the pathogenesis of this common hereditary deafness induced by connexin mutations.

Flow paths of plant tissue residues and digesta through gastrointestinal segments in Spanish goats and methodological considerations.

A sequence of eight twice-daily meals, each marked with different rare earth elements, was fed to 24 Spanish goats (BW = 20.6 +/- 1.94 kg) to produce meal-based profiles of rare earth markers within segments of the gastrointestinal digesta on subsequent slaughter. Accumulative mean residence time and time delay of rare earths and segmental and accumulative mean residence times of indigestible NDF (IDF) were estimated for each sampled segment. Diets consisted of ad libitum access to bermudagrass hay with a limit feeding of one of four supplements: 1) minerals (basal, B); 2) B + energy (E); 3) B + CP (CP); or 4) B + E + CP for 84 d. Mean daily intake (g/kg of BW) during the 5 d before slaughter differed (P < 0.05) via diet for DM but not for IDF (8.0 +/- 0.35 g/kg of BW). Larger estimates of cumulative mean residence time for IDF vs. rare earths were suggested to be the consequence of a meal-induced bias in the single measurement of IDF pool size by anatomical site. The rare earth compartment method was considered more reliable than the IDF pool dilution method because it yielded flow estimates based on the flux of eight meal-dosed rare earth markers over 4 d and was independent of anatomical definitions of pool size. Statistically indistinguishable estimates for gastrointestinal mean residence times for IDF and rare earths conform to assumed indelibility for the specifically applied rare earths and indigestibility of IDF. The potentially digestible NDF (PDF):IDF ratio of dietary fragments (0.8) progressively decreased in the following order: caudodorsal reticulorumen (0.390) > crainodorsal reticulorumen (0.357) approximately reticulum (0.354) > mid-dorsal reticulorumen (0.291) approximately ventral reticulorumen (0.286), to that within the omasal folds and in the abomasum (0.259). Such a gradient of progressively aging mixture of plant tissue fragments is consistent with age-dependent flow paths established in the reticulorumen and flowing to the omasum and abomasum. Such heterogeneity of fragment ages within the reticulorumen is also indicated by the superior fit of marker dose site double dagger marker sampling site model assumptions. Additionally, cyclic meal- and rumination-induced variations in escape rate occur. Estimates of mean escape rates over days, needed for the practice of ruminant nutrition, must consider the complex interactions among plant tissues and the dynamics of their ruminal digestion of PDF.

Loss-of-function and residual channel activity of connexin26 mutations associated with non-syndromic deafness.

Connexins are the protein subunits of gap junction channels that allow a direct signaling pathway between networks of cells. The specific role of connexin channels in the homeostasis of different organs has been validated by the association of mutations in several human connexins with a variety of genetic diseases. Several connexins are present in the mammalian cochlea and at least four of them have been proposed as genes causing sensorineural hearing loss. We have started our functional analysis by selecting nine mutations in Cx26 that are associated with non-syndromic recessive deafness (DFNB1). We have observed that both human Cx26 wild-type (HCx26wt) and the F83L polymorphism, found in unaffected controls, generated electrical conductance between paired Xenopus oocytes, which was several orders of magnitude greater than that measured in water-injected controls. In contrast, most recessive Cx26 mutations (identified in DFNB1 patients) resulted in a simple loss of channel activity. In addition, the V37I mutation, originally identified as a polymorphism in heterozygous unaffected individuals, was devoid of function and thus may be pathologically significant. Unexpectedly, we have found that the recessive mutation V84L retained functional activity in both paired Xenopus oocytes and transfected HeLa cells. Furthermore, both the magnitude of macroscopic junctional conductance and its voltage-gating properties were indistinguishable from those of HCx26wt. The identification of functional differences of disease causing mutations may lead to define which permeation or gating properties of Cx26 are necessary for normal auditory function in humans and will be instrumental in identifying the molecular steps leading to DFNB1.

Prenatal lens development in connexin43 and connexin50 double knockout mice.

PURPOSE: To determine the roles of intercellular communication in embryonic eye growth and development, mice with a targeted deletion of the Cx43 gene were examined, and mice without both Cx43 and Cx50 were generated and analyzed. METHODS: Embryonic eyes and lenses from wild-type mice, or mice deficient in Cx43, Cx50, or both Cx43 and Cx50 were collected and analyzed structurally by light and electron microscopy, immunohistochemically using connexin-specific antibodies, biochemically by Western blot analysis, and physiologically by measuring patterns of junctional communication revealed by iontophoretic injection of junction-permeable reporter molecules. RESULTS: Cx50 expression was limited to the ocular lens and was not detected in either the cornea or the retina. Cx43(-/-) embryos showed development of structurally normal lenses and eyes when examined by light and electron microscopy through embryonic day (E)18.5. In addition, Cx43(-/-) lenses synthesized four different markers of lens differentiation: MIP26, alphaA-crystallin, alphaB-crystallin, and gamma-crystallin. Double-knockout lenses were also histologically normal through E18.5 and synthesized the four lens differentiation markers. When assayed by intracellular injection with Lucifer yellow (Molecular Probes, Eugene, OR) and neurobiotin at E15.5, Cx43(-/-)/Cx50(-/-) lenses retained gap junction-mediated dye transfer between fiber cells. In contrast, dye transfer in double-knockout lenses was dramatically reduced between epithelial cells and was eliminated between epithelial cells and fibers. CONCLUSIONS: These data indicate that the unique functional properties of both Cx43 and Cx50 are not required for prenatal lens development and that connexin diversity is required for regulation of postnatal growth and homeostasis.

trans-dominant inhibition of connexin-43 by mutant connexin-26: implications for dominant connexin disorders affecting epidermal differentiation.

Dominant mutations of GJB2-encoding connexin-26 (Cx26) have pleiotropic effects, causing either hearing impairment (HI) alone or in association with palmoplantar keratoderma (PPK/HI). We examined a British family with the latter phenotype and identified a new dominant GJB2 mutation predicted to eliminate the amino acid residue E42 (DeltaE42) in Cx26. To dissect the pathomechanisms that result in diverse phenotypes of dominant GJB2 mutations, we studied the effect of three Cx26 mutants (DeltaE42, D66H and R75W) identified in individuals with PPK/HI, and another (W44C) present in individuals with non-syndromic HI on gap junctional intercellular communication. We expressed mutant Cx26 alone and together with the epidermal connexins Cx26, Cx37 and Cx43 in paired Xenopus oocytes, and measured the intercellular coupling by dual voltage clamping. Homotypic expression of each connexin as well as co-expression of wild-type (wt) Cx26/wtCx43 and wtCx26/wtCx37 yielded variable, yet robust, levels of channel activity. However, all four Cx26 mutants were functionally impaired and failed to induce intercellular coupling. When co-expressed with wtCx26, all four mutants suppressed the wtCx26 channel activity consistent with a dominant inhibitory effect. However, only those Cx26 mutants associated with a skin phenotype also significantly (P<0.05) inhibited intercellular conductance of co-expressed wtCx43, indicating a direct interaction of mutant Cx26 units with wtCx43. These results demonstrate, for the first time, a trans-dominant negative effect of Cx26 mutants in vitro. Furthermore, they support a novel concept suggesting that the principal mechanism for manifestation of dominant GJB2 mutations in the skin is their dominant interference with the function of wtCx43. This assumption is further corroborated by our finding that Cx26 and Cx43 focally colocalize at gap junctional plaques in affected skin tissue of two carriers of DeltaE42.

Molecular cloning, expression analysis, and functional characterization of connexin44.1: a zebrafish lens gap junction protein.

The connexin family of genes codes for proteins that oligomerize into a connexon of six subunits to form one half of the gap junction channel. Gap junctions are plasma membrane structures that mediate intercellular communication by joining the cytoplasm of two cells, allowing the passage of small molecules and metabolites, and contributing significantly to the maintenance of tissue homeostasis. The signaling mediated by these junctions appears to be necessary for the correct timing of key developmental events. This communication is especially important in the avascular lens where the intercellular passage of metabolites, second messengers, and ions is necessary to maintain the correct ionic balance in the lens fibre cells, and prevent cataract formation. To characterize the role that the connexin genes play in development, a novel connexin was cloned from zebrafish. A genomic clone was isolated that contained a 1,173 base open reading frame. The nucleotide sequence in this open reading frame shows extensive sequence similarity to mouse connexin50 (Cx50), chicken Cx45.6, sheep Cx49, and human Cx50. The protein encoded by this open reading frame contains 391 amino acids, with a predicted molecular weight of 44.1 kDa and a typical connexin transmembrane topology. By using the LN54 radiation hybrid panel, the Cx44.1 gene was mapped to linkage group 1. Whole-mount in situ hybridization and Northern blot analyses were performed on zebrafish embryos at various developmental stages to characterize the developmental expression of the Cx44.1 message. The ocular lens was the only tissue in which Cx44.1 transcripts were detected. The transcripts were first detected in the lens around 24 hr post fertilization and remained detectable until 120 hr post fertilization. Electrophysiological analysis of Cx44.1 channels revealed gating properties that were virtually identical to the mouse and chicken orthologues of Cx44.1.

Influence of dietary carnitine in growing sheep fed diets containing non-protein nitrogen.

The influence of supplemental L-carnitine was investigated in growing sheep fed rations containing non-protein nitrogen (NPN). The experiment was conducted as a randomized block design with a 2x2 factorial arrangement of treatments. Lambs (77.4kg BW, n=24) were fed a total mixed ration (12.1-13.6% CP) with two levels of L-carnitine (0 or 250ppm) and two levels of NPN (urea contributing 0 or 50% of total dietary N) for a 50-day period. Jugular blood samples were collected at 0, 1, and 3h post-feeding, and ruminal fluid samples were collected at 1h post-feeding, during days 1, 8, 29, and 50 of the experiment. Average daily gain (121 versus 214g) was lower (P<0.0001) in lambs fed the NPN diets. Lambs consuming diets containing NPN had higher (P<0.0001) ruminal fluid pH (6.6 versus 5.9), ruminal ammonia N (4.8 versus 2.8mmol/l), and plasma ammonia N (177.1 versus 49.5µmol/l) than lambs not fed NPN. Additionally, lambs fed the NPN diets had lower plasma urea N (14.5 versus 17.5mmol/l; P<0.003) and thyroxine (T(4)) concentrations (65.8 versus 78.4ng/ml; P<0.02), and lower T(4):triiodothyronine (T(3)) ratio (37.9 versus 43.9; P<0.02). Plasma glucose concentrations were higher (P<0.05) in lambs fed L-carnitine (3.83 versus 3.70mmol/l). Two oral urea load tests (OULT 1 and OULT 2) were conducted during the 50-day trial. Urea solutions (0.835g/kg(0.75) BW) were administered as oral drenches. During the OULT 1 (day 10), plasma ammonia N and glucose concentrations were highest (P<0.0001) in the lambs fed NPN with L-carnitine compared with lambs fed control, L-carnitine, and NPN diets. During the OULT 2 (day 50), plasma ammonia N was highest (P<0.0001) in the NPN and NPN with L-carnitine groups compared with the control and L-carnitine groups. Plasma glucose was lowest (P<0.04) in the NPN with L-carnitine group compared with the NPN and L-carnitine groups, but did not differ (P>0.10) from the control group. Plasma urea N levels in both OULT 1 and OULT 2 were lower (P<0.0001) in the NPN and NPN with L-carnitine groups compared with the control and L-carnitine groups. In the present experiment, production and plasma criteria were affected by NPN incorporation in the diets. Production criteria were not affected by inclusion of L-carnitine in the diet, however, L-carnitine reduced experimentally induced hyperammonemia by day 50 of the trial.

Identification and functional expression of HCx31.9, a novel gap junction gene.

By combining in silico and bench molecular biology methods we have identified a novel human gap junction gene that encodes a protein designated HCx31.9. We have determined its human chromosomal location and gene structure, and we have identified a putative mouse ortholog, mCx30.2. We have observed the presence of HCx31.9 in human cerebral cortex, liver, heart, spleen, lung, and kidney and the presence of mCx30.2 in mouse cerebral cortex, liver and lung. Moreover, preliminary data on the electrophysiological properties of HCx31.9 have been obtained by functional expression in paired Xenopus oocytes and in transfected N2A cells.

Functional analysis of a dominant mutation of human connexin26 associated with nonsyndromic deafness.

Cx26 has been implicated in dominant (DFNA3) and recessive (DFNB1) forms of nonsyndromic sensorineural deafness. While most homozygous DFNB1 Cx26 mutations result in a simple loss of channel activity, it is less clear how heterozygous mutations in Cx26 linked to DFNA3 cause hearing loss. We have tested the ability of one dominant mutation (W44C) to interfere with wild-type human Cx26 (HCx26wt). HCx26wt induced robust electrical conductance between paired oocytes, and facilitated dye transfer between transfected HeLa cells. In contrast, oocyte pairs injected with only W44C were not electrically coupled above background levels, and W44C failed to dye couple transfected HeLa cells. Moreover, W44C dramatically inhibited intercellular conductance of HCx26wt when co-expressed in an equal ratio, and the low levels of residual conductance displayed altered gating properties. A nonfunctional recessive mutation (W77R) did not inhibit the ability of HCx26wt to form functional channels when co-injected in the same oocyte pairs, nor did it alter HCx26wt gating. These results provide evidence for a functional dominant negative effect of the W44C mutant on HCx26wt and explain how heterozygous Cx26 mutations could contribute to autosomal dominant deafness, by resulting in a net loss, and/or alteration, of Cx26 function.

Gap junctions: fates worse than death?

The reasons for the molecular heterogeneity of connexin channels in vivo remain unclear. Functional replacement of one connexin gene with another has now revealed unexpected phenotypes and shows that cellular homeostasis depends not simply on cell-cell communication but also on the correct types of connexin.

Intercellular communication in the eye: clarifying the need for connexin diversity.

In the vertebrate eye, virtually every cell type is directly coupled to its neighbors by intercellular channels present in gap junctions. Although these structures share the common property of allowing adjacent cells to directly exchange ions, second messengers and small metabolites, intercellular channels in the eye also play a specific role in distinct functions such as neuronal transmission at electrotonic synapses in the retina, and the maintenance of homeostasis in the avascular lens. The structural proteins comprising these channels, the connexins (Cx), are a multigene family of which many members are expressed in the eye, even in the same cell type. This molecular heterogeneity poses the crucial question of whether and how a diversity in gap junctional structural proteins influences intercellular communication in ocular tissues. This review will focus on two recent advances in the understanding of connexin diversity in regard to the eye. First, connexin knockouts have demonstrated that postnatal development and homeostasis in the lens requires multiple connexin proteins. Secondly, functional characterization of new connexins that are abundantly expressed in the retina has revealed biophysical properties that mimic those recorded from retinal neurons.

Functional analysis of human Cx26 mutations associated with deafness.

Mutations in the connexin26 (Cx26) gene are not only a major cause of nonsyndromic deafness, but can also cause syndromic forms of hearing loss that are associated with palmoplantar keratoderma (PPK, i.e., Vohwinkel's syndrome). It is not clear how two very distinct pathologies can arise from different mutations within the same connexin gene. This review summarizes the available data on wildtype and mutant Cx26 channel behavior that has been obtained in the paired Xenopus oocyte assay. These results suggest that dominant and recessive loss of function mutations in Cx26 can cause nonsyndromic deafness, but cannot easily explain the syndromic forms exhibiting PPK. Dominant Cx26 mutations that can cause both PPK and deafness must show some additional alteration of function beyond a simple inhibition of Cx26 activity.

Functional properties, developmental regulation, and chromosomal localization of murine connexin36, a gap-junctional protein expressed preferentially in retina and brain.

Retinal neurons of virtually every type are coupled by gap-junctional channels whose pharmacological and gating properties have been studied extensively. We have begun to identify the molecular composition and functional properties of the connexins that form these 'electrical synapses,' and have cloned several that constitute a new subclass (gamma) of the connexin family expressed predominantly in retina and brain. In this paper, we present a series of experiments characterizing connexin36 (Cx36), a member of the gamma subclass that was cloned from a mouse retinal cDNA library. Cx36 has been localized to mouse chromosome 2, in a region syntenic to human chromosome 5, and immunocytochemistry showed strong labeling in the ganglion cell and inner nuclear layers of the mouse retina. Comparison of the developmental time course of Cx36 expression in mouse retina with the genesis of the various classes of retinal cells suggests that the expression of Cx36 occurs primarily after cellular differentiation is complete. Because photic stimulation can affect the gap-junctional coupling between retinal neurons, we determined whether lighting conditions might influence the steady state levels of Cx36 transcript in the mouse retina. Steady-state levels of Cx36 transcript were significantly higher in animals reared under typical cyclic-light conditions; exposure either to constant darkness or to continuous illumination reduced the steady-state level of mRNA approximately 40%. Injection of Cx36 cRNA into pairs of Xenopus oocytes induced intercellular conductances that were relatively insensitive to transjunctional voltage, a property shared with other members of the gamma subclass of connexins. Like skate Cx35, mouse Cx36 was unable to form heterotypic gap-junctional channels when paired with two other rodent connexins. In addition, mouse Cx36 failed to form voltage-activated hemichannels, whereas both skate and perch Cx35 displayed quinine-sensitive hemichannel activity. The conservation of intercellular channel gating contrasts with the failure of Cx36 to make hemichannels, suggesting that the voltage-gating mechanisms of hemichannels may be distinct from those of intact intercellular channels.

Innexin-3 forms connexin-like intercellular channels.

Innexins comprise a large family of genes that are believed to encode invertebrate gap junction channel-forming proteins. However, only two Drosophila innexins have been directly tested for the ability to form intercellular channels and only one of those was active. Here we tested the ability of Caenorhabditis elegans family members INX-3 and EAT-5 to form intercellular channels between paired Xenopus oocytes. We show that expression of INX-3 but not EAT-5, induces electrical coupling between the oocyte pairs. In addition, analysis of INX-3 voltage and pH gating reveals a striking degree of conservation in the functional properties of connexin and innnexin channels. These data strongly support the idea that innexin genes encode intercellular channels.

Dietary protein and chromium tripicolinate in Suffolk wether lambs: effects on production characteristics, metabolic and hormonal responses, and immune status.

Thirty-two Suffolk wether lambs were fed for 84 d in a 2 x 2 factorial experiment using two levels of dietary protein (9.0 to 12.1% CP, low protein, LP; or 12.8 to 14.4% CP, high protein, HP) and supplemental Cr (none, C; or 400 ppb Cr as chromium tripicolinate, Cr). At 14- to 21-d intervals, lambs were weighed, and jugular blood samples were collected. Mean ADG and carcass weight (P > .10) did not differ. In lambs fed HP, Cr reduced liver weight and increased kidney weight (P < .01). Lambs fed HP had elevated plasma urea N (PUN; P < .01) and albumin (P < .04). During an i.v. epinephrine challenge on d 43, plasma cortisol declined in lambs fed Cr (Cr x time, P < .03) and in lambs fed LP (CP x time, P < .001). An i.v. glucose tolerance test conducted 3 h later showed that supplemental Cr decreased glucose clearance rate in lambs fed HP (CP x Cr, P < .10) but not in lambs fed LP. On d 62, PUN was increased in lambs fed HP (P < .001) between 0 and 3 h postprandial, and there was a Cr x CP interaction (P < .04). Postprandial plasma NEFA declined with Cr vs C (Cr x time, P < .07) and with HP vs LP (CP x time, P < .10). By d 66, lambs fed Cr had an elevated (P < .03) blood platelet and fibrinogen content. Chromium increased erythrocyte count in lambs fed HP (Cr x CP, P < .08), and isolated peripheral lymphocytes had greater blastogenic response to 4 microg/mL of phytohemagglutinin (Cr x CP, P < .001). The lymphocyte response to pokeweed mitogen (.2 microg/mL) was reduced in lambs fed Cr (P < .10). In the present experiment, Cr supplementation had minimal and inconsistent effects on production and metabolic criteria of lambs.

Functional characteristics of skate connexin35, a member of the gamma subfamily of connexins expressed in the vertebrate retina.

Retinal neurons are coupled by electrical synapses that have been studied extensively in situ and in isolated cell pairs. Although many unique gating properties have been identified, the connexin composition of retinal gap junctions is not well defined. We have functionally characterized connexin35 (Cx35), a recently cloned connexin belonging to the gamma subgroup expressed in the skate retina, and compared its biophysical properties with those obtained from electrically coupled retinal cells. Injection of Cx35 RNA into pairs of Xenopus oocytes induced intercellular conductances that were voltage-gated at transjunctional potentials >/= 60 mV, and that were also closed by intracellular acidification. In contrast, Cx35 was unable to functionally interact with rodent connexins from the alpha or beta subfamilies. Voltage-activated hemichannel currents were also observed in single oocytes expressing Cx35, and superfusing these oocytes with medium containing 100 microm quinine resulted in a 1.8-fold increase in the magnitude of the outward currents, but did not change the threshold of voltage activation (membrane potential = +20 mV). Cx35 intercellular channels between paired oocytes were insensitive to quinine treatment. Both hemichannel activity and its modulation by quinine were seen previously in recordings from isolated skate horizontal cells. Voltage-activated currents of Cx46 hemichannels were also enhanced 1. 6-fold following quinine treatment, whereas Cx43-injected oocytes showed no hemichannel activity in the presence, or absence, of quinine. Although the cellular localization of Cx35 is unknown, the functional characteristics of Cx35 in Xenopus oocytes are consistent with the hemichannel and intercellular channel properties of skate horizontal cells.

Genetic diseases and gene knockouts reveal diverse connexin functions.

Intercellular channels present in gap junctions allow cells to share small molecules and thus coordinate a wide range of behaviors. Remarkably, although junctions provide similar functions in all multicellular organisms, vertebrates and invertebrates use unrelated gene families to encode these channels. The recent identification of the invertebrate innexin family opens up powerful genetic systems to studies of intercellular communication. At the same time, new information on the physiological roles of vertebrate connexins has emerged from genetic studies. Mutations in connexin genes underlie a variety of human diseases, including deafness, demyelinating neuropathies, and lens cataracts. In addition, gene targeting of connexins in mice has provided new insights into connexin function and the significance of connexin diversity.

Influence of intravenous L-carnitine administration in sheep preceding an oral urea drench.

Two experiments were conducted to investigate the effect of i.v. administration of L-carnitine on selected metabolites in sheep and to determine the feasibility of using L-carnitine to ameliorate the deleterious effects of hyperammonemia in sheep. In Exp. 1, i.v. L-carnitine solutions were administered at three levels in a replicated Latin square: 0 (CONT), 6.36 (CAR 1), and 12.72 (CAR 2) mmol L-carnitine/kg x (75) BW using Suffolk ewes (n = 6; average BW 75+/-3 kg). Plasma L-carnitine concentration was increased (P<.05) by treatment (51.9 vs 102.3, and 96.4 micromol/L in CONT, CAR 1, and CAR 2, respectively). Plasma glucose concentration was elevated (P<.05) in CAR 2 and CAR 1. Plasma NEFA concentration was highest (P<.05) in CAR 2. Area under the response curve for glucose was greater (P<.02) in CAR 2. In Exp. 2, Suffolk ewes (n = 16; average BW 48+/-2 kg) were used in a randomized complete block design with a 2x2 factorial treatment arrangement to determine the effects of i.v. L-carnitine administration during an oral urea load test (OULT). L-Carnitine (0 and 6.36 mmol/kg x (75) BW) was administered i.v. at 30 min, and an oral urea drench (50% wt/vol; 0 and 300 mg/kg BW) was administered at 60 min. Plasma L-carnitine was increased (P<.0001) by i.v. L-carnitine. Plasma ammonia N was highest (P<.0001) in the UREA treatment compared with the CONT, CARN, and CARN + UREA treatments (148 vs 95, 101, and 108 micromol/L, respectively). Intravenous L-carnitine administration influenced plasma glucose and NEFA concentrations in sheep and, when administered 30 min preceding an OULT, prevented the development of subclinical hyperammonemia in sheep.