Novel rapid method for the characterisation of polymeric sugars from macroalgae.

Laminarins are storage polysaccharides found only in brown seaweeds, specifically Laminarialaes and Fucales. Laminarin has been shown to have anti-apoptotic and anti-tumoural activities and is considered as a nutraceutical component that can positively influence human health. The structure is species dependent, generally composed of linear ß(1-3) glucans with intrachain ß(1-6) branching and varies according to harvest season and environmental factors. Current methods for analysis of molar mass and DP length are technically demanding and are not widely available. Here, we present a simple inexpensive method which enables rapid analysis of laminarins from macroalgal biomass using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) without the need for hydrolysis or further processing. This is based on the linear relationship observed between log10 DP and retention time following separation of laminarins on a CarboPac PA-100 column (Dionex) using standard 1,3-ß-d-gluco-oligosaccharides ranging in DP from 2 to 8. This method was applied to analyse laminarin oligomers in extracts from different species harvested from within the intertidal zone on Welsh rocky shores containing laminarin polymers with different ranges of DP. The degree of polymerisation and extrapolated molar mass agreed well with values estimated by LC-ESI/MS n analysis and those reported in the literature.

Fibronectin non-amyloid glomerulopathy.

A 41-year-old Burmese man presented with nephrotic syndrome, a creatinine level of 150 micromol/L and limited clinical history. His renal biopsy demonstrated glomerulopathy with large eosinophilic deposits in the mesangium and capillary loops that were negative for Congo red, slightly positive for periodic acid-Schiff and blue with Masson trichrome stain. Immunofluorescence microscopy with a routine antibody panel was unhelpful. Electron microscopy demonstrated extensive, moderately electron-dense deposits in the subendothelial space, subepithelial space and mesangium that could be differentiated from adjacent basement membrane by their increased electron density. The deposits contained finely granular material and occasional filaments with variable diameter ranging from 9-16 nm. Fibronectin glomerulopathy was suspected from anti-fibronectin immunohistochemistry that showed positive staining of thickened capillary loops. This staining was subsequently confirmed by immunoelectron microscopy demonstrating the presence of cellular fibronectin (cFN) in deposits. Significantly, deposition of fibronectin appeared to have occurred in the absence of thickening or folding of the adjacent basement membrane. The prominent mesangial location of deposits containing a cFN isotype may indicate that retention of local fibronectin produced in the mesangium has contributed to this pathology.

Induction of passive Heymann nephritis in complement component 6-deficient PVG rats.

Passive Heymann nephritis (PHN), a model of human membranous nephritis, is induced in susceptible rat strains by injection of heterologous antisera to rat renal tubular Ag extract. PHN is currently considered the archetypal complement-dependent form of nephritis, with the proteinuria resulting from sublytic glomerular epithelial cell injury induced by the complement membrane attack complex (MAC) of C5b-9. This study examined whether C6 and MAC are essential to the development of proteinuria in PHN by comparing the effect of injection of anti-Fx1A antisera into PVG rats deficient in C6 (PVG/C6(-)) and normal PVG rats (PVG/c). PVG/c and PVG/C6(-) rats developed similar levels of proteinuria at 3, 7, 14, and 28 days following injection of antisera. Isolated whole glomeruli showed similar deposition of rat Ig and C3 staining in PVG/c and PVG/C6(-) rats. C9 deposition was abundant in PVG/c but was not detected in PVG/C6(-) glomeruli, indicating C5b-9/MAC had not formed in PVG/C6(-) rats. There was also no difference in the glomerular cellular infiltrate of T cells and macrophages nor the size of glomerular basement membrane deposits measured on electron micrographs. To examine whether T cells effect injury, rats were depleted of CD8+ T cells which did not affect proteinuria in the early heterologous phase but prevented the increase in proteinuria associated with the later autologous phase. These studies showed proteinuria in PHN occurs without MAC and that other mechanisms, such as immune complex size, early complement components, CD4+ and CD8+ T cells, disrupt glomerular integrity and lead to proteinuria.

Severe intimal hyperplasia with ischaemic colitis in two patients with hepatitis C.

Many extrahepatic manifestations of hepatitis C have been described, including renal disease and vasculitis. We describe the novel finding of intimal hyperplasia associated with severe ischaemic events in two patients with hepatitis C. The combination of genotype 1a hepatitis C virus, rapidly progressive mesangiocapillary glomerulonephritis and intimal hyperplasia with ischaemic sequelae, may represent a new syndrome.

A voltage- and Ca2+-dependent big conductance K channel in cochlear spiral ligament fibrocytes.

Evidence is accruing that spiral ligament fibrocytes (SLFs) play an important role in cochlear K(+) homeostasis, but little direct physiological data is available to support this concept. Here we report the presence and characterization of a voltage- and Ca(2+)-dependent big-conductance K (BK) channel in type I SLFs cultured from the gerbil cochlea. A single-channel conductance of 298+/-5.6 pS (n=28) was measured under symmetrical K(+). Membrane potentials for half-maximal open probability (P(o)) were -67, -45 and 85 mV with cytosolic free-Ca(2+) levels of 0.7 mM, 10 microM and 1 microM, respectively (n=8-14). The Hill coefficient for Ca(2+) affinity was 1.9 at a membrane potential of 60 mV (n=6). The BK channel showed very low activity (P(o)=0.0019, n=5) under normal physiological conditions, suggesting a low resting intracellular free [Ca(2+)]. Pharmacological results fit well with the profile of classic BK channels. The estimated half-maximal inhibitory concentration and Hill coefficient for tetraethylammonium were 0.086+/-0.021 mM and 0.99, respectively (n=4-9). In whole cell recordings, the voltage-activated outward K current was inhibited 85.7+/-4.5% (n=6) by 0.1 microM iberiotoxin. A steady-state kinetic model with two open and two closed stages best described the BK gating process (tau(o1) 0.23+/-0.08 ms, tau(o2) 1.40+/-0.32 ms; tau(c1) 0.26+/-0.09 ms, tau(c2) 3.10+/-1.2 ms; n=11). RT-PCR analyses revealed a splice variant of the BK channel alpha subunit in cultured type I SLFs and freshly isolated spiral ligament tissues. The BK channel is likely to play a major role in regulating the membrane potential of type I SLFs, which may in turn influence K(+) recycling dynamics in the mammalian cochlea.

Effects of gap junction uncoupling in the gerbil cochlea.

OBJECTIVE: To gain insight into molecular and cellular mechanisms regulating cochlear potassium (K+) recycling, including the possible effects of mutations in the gene, which encodes the gap junction protein connexin 26. Intercellular K+ flux was manipulated in vivo by infusion of the gap junction uncoupler proadifen (SKF-525A) into perilymph. Functional and structural alterations induced by gap junction blockade were assessed by electrophysiological and morphologic analysis. STUDY DESIGN: Laboratory study using an animal model. METHODS: Physiological effects of acute and chronic uncoupling of gap junctions in the Mongolian gerbil inner ear were evaluated by measurement of compound action potential (CAP) thresholds and input-output (I/O) functions, distortion product otoacoustic emissions (DPOAE), and the endocochlear potential (EP). Morphologic changes were assessed by electron microscopy. RESULTS: Acute exposures to proadifen resulted in large decreases in EP values, DPOAE magnitudes, and CAP I/O maximum amplitudes and an increase in high-frequency CAP thresholds. These physiological changes were accompanied by vacuolization of type II and type V fibrocytes in the lateral wall of the cochlea. Chronic treatments revealed some recovery in EP values and CAP thresholds, which showed a relatively flat 15- to 20-dB elevation across frequencies. CONCLUSIONS: Gap junctions play a significant role in normal cochlear function. In particular they appear to be essential for maintaining the EP, an activity that could be related to their participation in K+ recycling. Thus, hearing losses associated with mutations in the gene that alter the expression or function of connexin 26 may result from a diminished capacity to recycle K+ from perilymph back to the stria vascularis and a consequent decline in the EP.

Attenuation of experimental allergic encephalomyelitis in complement component 6-deficient rats is associated with reduced complement C9 deposition, P-selectin expression, and cellular infiltrate in spinal cords.

The role of Ab deposition and complement activation, especially the membrane attack complex (MAC), in the mediation of injury in experimental allergic encephalomyelitis (EAE) is not resolved. The course of active EAE in normal PVG rats was compared with that in PVG rats deficient in the C6 component of complement (PVG/C6(-)) that are unable to form MAC. Following immunization with myelin basic protein, PVG/C6(-) rats developed significantly milder EAE than PVG/C rats. The anti-myelin basic protein response was similar in both strains, as was deposition of C3 in spinal cord. C9 was detected in PVG/C rats but not in PVG/C6(-), consistent with their lack of C6 and inability to form MAC. In PVG/C6(-) rats, the T cell and macrophage infiltrate in the spinal cord was also significantly less than in normal PVG/C rats. There was also reduced expression of P-selectin on endothelial cells, which may have contributed to the reduced cellular infiltrate by limiting migration from the circulation. Assay of cytokine mRNA by RT-PCR in the spinal cords showed no differences in the profile of Th1 or Th2 cytokines between PVG/C and PVG/C6(-) rats. PVG/C rats also had a greater increase in peripheral blood white blood cell, neutrophil, and basophil counts than was observed in the PVG/C6(-). These findings suggest that the MAC may have a role in the pathogenesis of EAE, not only by Ig-activated MAC injury but also via induction of P-selectin on vascular endothelium to promote infiltration of T cells and macrophages into the spinal cord.

Developmental expression of monocarboxylate transporter in the gerbil inner ear.

The expression of H+-monocarboxylate cotransporters (MCTs) that facilitate cell uptake of lactate, pyruvate and other monocarboxylates was investigated in the adult and postnatally developing gerbil inner ear. In the mature cochlea, immunoreactive MCT1 was present in marginal cells of the stria vascularis and in type II, suprastrial and limbal fibrocytes. In the adult vestibular system, dark cells and a subpopulation of fibrocytes immediately underlying maculae and cristae stained strongly for MCT1. Satellite cells surrounding mature spiral and vestibular ganglia neurons also expressed MCT1. MCT1 immunoreactivity was present at birth in marginal and dark cells, at 8 days after birth in fibrocytes and at 12 days after birth in satellite cells, and coincided precisely with the developmental expression of Na,K-ATPase in these sites. The coexpression of MCT1 and Na,K-ATPase in these cell types points to MCT1 as an important source of energy to drive inner ear Na,K-ATPase activity. In the adult inner ear, MCT2 was detectable only in tectal cells of the cochlea and supporting cells of the crista ampullaris. Immunostaining was first observed at 16 days after birth in tectal and at 20 days after birth in supporting cells, and at the same time immunoreactive aquaporin 4 appeared in these cells. The coexpression of MCT2 and aquaporin 4 suggests a possible role for MCT2 in regulating transcellular water movement. Because MCT2 facilitates the transport of acidic intermediates, its biological significance also could relate to modulation of cell pH and volume. Maintenance of the inner ear's unique ion and fluid gradients is essential to normal hearing and balance and requires the expenditure of large amounts of energy. The cellular distribution of MCT1 and MCT2 points to their participation in generating these electrochemical gradients and their potential involvement in sensory deficits associated with various inner ear disorders.

Il-4 therapy prevents the development of proteinuria in active Heymann nephritis by inhibition of Tc1 cells.

The role of IL-4, a key Th2 cytokine, in promoting or inhibiting active Heymann nephritis (HN) was examined. HN is induced by immunization with Fx1A in CFA, and proteinuria in HN is associated with subepithelial IgG and C3 deposition and infiltration of CD8(+) T-cytotoxic 1 (Tc1) cells and macrophages into glomeruli, as well as induction of Abs to Crry. Treatment with rIL-4 from the time of Fx1A/CFA immunization stimulated an earlier IgG1 response to Fx1A, induced anti-Crry Abs, and up-regulated IL-4 mRNA in lymphoid tissue, but did not alter proteinuria. Treatment with MRCOx-81, an IL-4-blocking mAb, resulted in greater proteinuria, which suggests endogenous IL-4 regulated the autoimmune response. Delay of rIL-4 treatment until 4 wk post-Fx1A/CFA immunization and just before the onset of proteinuria prevented the development of proteinuria and reduced Tc1 cell infiltrate in glomeruli. Delayed treatment with IL-4 had no effect on titer or isotype of Abs to Fx1A or on Ig, C3, and C9 accumulation in glomeruli. Treatment with rIL-13, a cytokine that alters macrophage function such as rIL-4, but has no direct effect on T or B cell function, reduced glomerular macrophage infiltrate, but did not prevent proteinuria or CD8+ T cell infiltrate. Anti-Crry Abs were paradoxically only induced with rIL-4 therapy, not in HN controls with proteinuria. It was concluded that the rIL-4 effect was probably by inhibition of Tc1 cells, which normally mediate the glomerular injury that results in proteinuria.

Diagnosis of kidney transplant obstruction using Mag3 diuretic renography.

Kidney transplant obstruction (KTO) following renal transplantation remains an important reversible cause of allograft dysfunction, requiring prompt diagnosis to prevent long-term graft damage. Although ultrasound can accurately diagnose renal transplant hydronephrosis, it cannot be used to assess its functional significance. We prospectively assessed the utility of technetium-99m mercaptoacetyltriglycine (Tc99m MAG3) diuretic renography for the diagnosis of allograft KTO, using standard visual and quantitative parameters, as well as calculated renal output efficiency (OE), which has been postulated to improve diagnostic yield. From a cohort of 45 renal transplant patients, two subgroups were formed. The first group of transplant recipients (n = 21) with stable function and no obstruction was used to derive normal values for Tc99m MAG3 scans. A second group of transplant recipients with acute renal dysfunction in whom KTO was clinically suspected was used to test the diagnostic utility of these derived values (n = 43 scans). KTO was diagnosed independently of the MAG3 scans by a fall in the serum creatinine in response to renal pelvis urinary drainage. OE in 12 renal allografts with KTO was significantly reduced compared with 31 Tc99m MAG3 scans without KTO (59.6 +/- 18.9 vs. 81.6 +/- 5.4%, p < 0.001). In KTO, the mean time of isotope appearance in the bladder (time to bladder [TTB]) was extended compared with unobstructed allografts (7.9 +/- 4.1 vs. 3.6 +/- 1.5 min, p < 0.001). Measurement of OE significantly improved the accuracy of diuretic MAG3 renography in the diagnosis of renal allograft KTO, especially when supplemented by the TTB, parenchymal transit time and shape of the renogram curve. Ureteric obstruction of the kidney transplant can be diagnosed with an OE reduced to < 75% (sensitivity 92%, specificity 87%) and confirmed by isotope hold-up in the pelvicalyceal system. A normal or slowly declining renogram curve effectively excluded KTO (sensitivity of 96%, negative predictive value of 84%). A parenchymal transit time of > 5 min and a TTB of > 7 min both yielded a sensitvity of 92% and a specificity of 81%. In conclusion, MAG3 renography is a clinically useful investigation for the diagnosis of KTO.

Immunohistochemical localization of phospholipase C isozymes in mature and developing gerbil cochlea.

The possibility that phospholipase C contributes to intracellular signaling in the cochlea was investigated by immunostaining for eight different isoforms of the enzyme. In the mature gerbil cochlea, expression of the isozymes varied widely among different cell types. The phospholipase C-beta1 isoform was detected in inner and outer hair cells, and spiral ganglion neurons where it may participate in regulating Ca(2+) flux. The beta3 isozyme was expressed in epithelial cells thought to mediate lateral and medial circulation of potassium. The beta2 isozyme was present in border, inner phalangeal and Hensen cells, the stria vascularis, and suprastrial and supralimbal fibrocytes where it also may be involved in regulating ion transport activities. The phospholipase C-gamma isozymes were expressed in supporting cells, the stria vascularis, and certain fibrocytes where they possibly participate in activating tyrosine kinase and modulating ion conductances. The delta2 isoform was found in pillar, outer sulcus and strial marginal cells as well as spiral ganglion neurons and their radial processes. Documentation of changes in the expression pattern of phospholipase C isoforms during postnatal development and knowledge of their distribution in several positive control tissues provided further data for speculation about the biologic significance of the cochlear reactivity. The results demonstrate a wide diversity of isozyme distribution in the cochlea and suggest that the enzymes affect activities of various cochlear cell types in different ways.

Ultrastructural changes in the spiral limbus associated with carboplatin-induced ablation of inner hair cells.

Four months after the selective ablation of inner hair cells by carboplatin, the interdental cell epithelium exhibited dilated intercellular spaces and cytosolic vacuoles not seen in controls. In addition, the wide, often electron-lucent phalanges observed in the interdental cells of the normal chinchilla collapsed into a dense stratum that projected enlarged polypoid profiles into the limbal zone of the tectorial membrane. Carboplatin treatment also resulted in the restructuring of the tectorial membrane overlying the limbus. Changes in this membrane included a variable accumulation of the basal matrix, the rearrangement of intermediate lucent spaces, and the disappearance of a superimposed filamentous mesh. These three strata are, under normal conditions, apparently involved in events underlying tectorial membrane renewal. The post-carboplatin changes in the interdental cells and tectorial membrane occurred exclusively in the proposed medial pathway for K+ diffusion from inner hair cells and presumably resulted from a reduced flow of ions and fluid secondary to the ablation of these cells.

Simulation-based medical planning for cardiovascular disease: visualization system foundations.

A technique for visualizing computational models along with volumetric imaging data in a real-time, interactive, simulation-based medical planning system for cardiovascular disease treatment is described. This technique involves an ordered rendering of faceted geometry and volumetric image data. We have developed a software system based on this image-fusion technique that is capable of capturing and representing the inherent anatomic constraints of an individual patient. Such constraints must be represented accurately in a medical planning system to ensure the validity of a potential procedure. A hypothetical clinical scenario is described for which vascular treatment plans were constructed pre-operatively without reference to the physical anatomic structure. These models were later embedded into patient-specific diagnostic MRA scans to establish the anatomic context for physiologic observations.

Canalicular reticulum in vestibular hair cells.

A membrane limited system referred to as canalicular reticulum (CR) has been demonstrated in the apical cytosol of the cochlea's inner and outer hair cells. Similarities between cochlear and vestibular hair cells prompted investigation of the presence of CR in hair cells of the gerbil vestibular labyrinth. A method of fixation with glutaraldehyde followed by an osmium-ferrocyanide mixture demonstrated abundant CR in the apex of both type I and type II hair cells. The CR was closely associated with numerous Golgi zones in the apex of the vestibular hair cells, indicating its genesis from Golgi cisternae. Also preserved in upper cytosol were discrete complexes of mitochondria with granular reticulum. These complexes offered a possible site for generating the membrane in Golgi zones and CR. Single and parallel cisternae of granular reticulum were observed in the basal half of the hair cells together with numerous synaptic-like vesicles. These cisternae with their terminal blebbing and accompanying canaliculi were interpreted as novel structures mediating synaptic vesicle genesis in vestibular hair cells in a manner comparable to that postulated for cochlear inner hair cells.

Structural evidence for ion transport and tectorial membrane maintenance in the gerbil limbus.

Cells medial to the tunnel of Corti were examined to assess fine structural features relevant to their proposed role in cochlear K(+) homeostasis. A dense network of canaliculi referred to as canalicular reticulum (CR) resided in the foot body of inner pillar cells, where it bordered and could resorb ions released from inner radial and spiral nerves. Lateral interdental cells (IDCs) formed columns which connected the inner sulcus epithelium with the base of the tectorial membrane's (TM) middle zone. A spout-like neck in cells at the top of lateral IDC columns housed a dense concentration of CR which resembled that characteristic of ion transporting epithelia and appeared to be located here for transporting ions and fluid toward the TM. Clustered IDCs in the center of the limbus connected underlying limbal stroma with the TM's limbal zone and appeared capable of transporting ions from stroma to TM. Abundant CR in limbal stellate fibrocytes evidenced their capacity to transport ions and fluid, presumably from inner sulcus epithelium toward central IDCs. The most medial IDCs possibly function as the terminus of an ion cycling path from scala vestibuli to endolymph. Light fibrocytes situated between supralimbal fibrocytes and medial IDCs appeared to serve as a link in this pathway. The limbal zone of the TM overlying central IDCs consisted of three distinct regions which offered a structural basis for transformation of an amorphous matrix supplied by central IDCs into the protofibrils of the membrane's middle zone.

Distribution of IkappaB proteins in gastric mucosa and other organs of mouse and gerbil.

The NF-kappaB/IkappaB complex is a major transcription regulator of inflammatory and immune responses. Helicobacter pylori infection causes chronic inflammation in gastric mucosa by inducing dissociation of the inhibitory IkappaB protein from the complex with a resulting increased expression of interleukin (IL)-8. To clarify which of several known IkappaB proteins could be involved in this inflammatory response, we undertook immunohistochemical examination of normal mouse stomach as well as other murine tissues for comparison, using polyclonal antibodies specific for alpha-, beta-, gamma-, and in-isoforms of IkappaB. The results showed strong immunoreactivity for the alpha-isoform in parietal cells and for the beta-isoform in pit cells of the stomach, along with the presence of these proteins in various other sites. Comparative staining revealed a similar but not identical distribution of IkappaB proteins in the Mongolian gerbil, a rodent model for H. pylori infection. The findings suggest that the alpha- and beta-isoforms are dominant IkappaB proteins in gastric parietal and foveolar cells, respectively, and point to a role for these transcription regulators in modulating pathological responses in stomach and other organs. (J Histochem Cytochem 48:191-199, 2000)

Molecular characterization and immunohistochemical localization of IV(4)GalNAcGgOse(4)Cer: a naturally occurring novel neutral glycosphingolipid in bovine brain.

A pair of novel neutral glycosphingolipids (Ngsls) has been identified in bovine brain. Their mobilities on thin layer chromatography were slightly different from a standard pentaglycosylceramide (nLcOse(5)Cer from bovine erythrocytes). The compounds were purified to homogeneity by column chromatography. Their fatty acid and base compositions, their monosaccharide compositions and sugar linkage positions were determined by gas-liquid chromato-graphy/mass spectrometry. Carbohydrate sequence analy-sis by(1)H NMR spectroscopy and stepwise exoglyco-sidase digestion indicated the following pentaglycosyl structure for the oligosaccharide moiety of both Ngsls: GalNAcbeta1-4Galbeta1-3GalNAcbeta1-4Galbeta1-4Gl c. The two Ngsls (abbreviated as IV(4)GalNAcGgOse(4)Cer or GalNAc-GA1), differ in their ceramide compositions, having d18:0 and d18:1 sphingosine as their long chain bases. A monospecific polyclonal anti-GalNAc-GA1 antibody, prepared in rabbit and purified by affinity chromatography, stained the neurons of cerebral cortex and cerebellum including Purkinje cells in adult rat brain, indicating that the novel GalNAc-GA1 is associated with cerebellar and other neurons in vertebrate central nervous system.

Ablation of inner hair cells by carboplatin alters cells in the medial K(+) flow route and disrupts tectorial membrane.

The thesis that K(+) effluxing from inner hair cells (IHCs) cycles medially back to endolymph through inner sulcus and interdental cells (IDCs) was tested by comparing control chinchilla cochleas with those in which IHCs were selectively destroyed by carboplatin. By light microscopy inner sulcus cells appeared tall and nearly empty in control ears, but 4 months after the carboplatin treatment many showed vacuolization and shrinkage. Inner pillar cells also consistently developed abnormal vacuoles after carboplatin treatment. Control cochleas exhibited lateral columns and central clusters of IDCs which at their apex possessed expanded presumably hydrated phalanges. Four months after carboplatin, the IDC epithelium enclosed empty looking spaces and the apical phalangeal compartment collapsed into a thin, apparently dehydrated layer. This alteration was accompanied by changes in the tectorial membrane (TM) whereby the membrane's limbal zone thickened progressively to form a tall hollow mound in advanced lesions. The clear spaces in the epithelium and collapse of the phalanges are thought to reflect diminished flow of ions and fluid through IDCs. The accumulation of limbal TM supports the premise that IDCs secrete macromolecules for TM turnover as well as ions and fluid for promoting lateral migration of its precursor constituents. Occurring after ablation of IHCs by carboplatin, the changes in inner pillar, inner sulcus and IDCs and limbal TM can be viewed as a secondary effect of the interrupted ion efflux from IHCs and as further evidence that this effluent follows a medial route.

Novel membranous structures in apical and basal compartments of inner hair cells.

Postfixation with a ferrocyanide-osmium tetroxide solution preserved a dense network of canaliculi extending from the apical to the upper lateral plasma membrane in cochlear inner hair cells (IHCs). Numerous Golgi bodies intermingled with this apical canalicular reticulum (CR). Osmium-ferrocyanide treatment also disclosed several previously unreported structures below the IHC nucleus. The first consisted of stacks of six or eight and sets of three parallel cisternae of rough endoplasmic reticulum spanning between clustered mitochondria. Some parallel cisternae ended with segmentation where they contacted mitochondria, and others terminated by transforming into blebs or continuing into canaliculi. A second feature was comprised of a complex of segmented cisternae and branching canaliculi with clustered mitochondria. Branching minicanaliculi with associated vesicles neighbored the complexes. A fourth entity consisted of synaptic-like vesicles that largely filled the subnuclear cytosol and congregated at synapses. An additional infranuclear structure was composed of slender canaliculi that collected near or streamed to plasmalemma, often next to a synapse. A paradoxical absence of rough endoplasmic reticulum above and Golgi zones below the nucleus provided evidence of atypical mechanisms for generating the membrane in CR and forming synaptic vesicles. The observations offer the view that IHCs are compartmentalized into an apical mechanoreceptor half and a basal half that affects neurotransmission. The apical CR provided a possible structural basis for sequestering the K+ known to influx apically and for directing its diffusion to the site of known efflux across the lateral plasmalemma. The codistribution of parallel cisternae, canalicular-mitochondrial complexes, and synaptic-like vesicles, all of which are unique to IHCs, implicated the cisternae and complexes in the genesis of the vesicles.

Distribution of canalicular reticulum in Deiters cells and pillar cells of gerbil cochlea.

Postfixation with an osmium tetroxide-potassium ferrocyanide solution revealed in supporting cells in the organ of Corti a network of canaliculi termed canalicular reticulum (CR). In Deiters cells (DCs), the CR filled cytosol at the base of the phalanx and under plasmalemma apposed to either the outer hair cells' (HCs) basal surface or nerve terminals. From these locations the CR, accompanied by dense fibrillar substance, descended along microtubule bundles and terminated by surrounding the rosette complex in the apical cytosol. Canalicular profiles protruding from the reticulum penetrated the loose meshwork comprising the periphery of the rosette complex to contact at intervals branches of the dense trabeculae that make up the core of the complex. This arrangement disclosed a structural and presumably functional relationship between outer HCs and the CR and rosette complex. Inner pillar cells (PCs) exhibited moderately abundant to sparse profiles of CR interspersed between microtubule bundles of the microtubule stalk that connected head and foot regions. More elaborate CR extended as a network upward from the top of the microtubule stalk part was into the head body and downward into a conical expansion of the stalk at the base of the cell. Cytosol on the medial side of the basal microtubule expansion contained abundant CR which in conjunction with CR between basal microtubule bundles lay situated for possible uptake of ions or neurotransmitter released from numerous adjoining nerves. CR in outer PCs resembled that in inner PCs but appeared less prevalent in the head and foot regions and did not occur in cytosol beside the basal microtubule stalk. Characteristically small Golgi complexes accompanied the reticulum in DCs and were prevalent in the upper regions but absent in the mid and lower part of inner PCs. Short cisternae in the Golgi stacks associated with CR contrasted with the lengthier cisternae in the complexes infrequently observed in cytosol outside the microtubule stalk of inner PCs.