Khalid M. Khan

Calcium channel subunits in the mouse cochlea.

Abstract: Messages for subunits of voltage‐gated calcium channels were examined in the cochlea of the CBAJ mouse by PCR analysis. Total RNA was extracted from the auditory organs of 16–18‐day‐old animals. After reverse transcription, resulting cDNA was amplified by PCR with primers targeted to nucleotide sequences corresponding to 12 different calcium channel subunits. PCR products representing subunit gene expression were strongly and consistently amplified for α1C, α1D, α1E, α2δ, β1, β3, and β4 but not for α1A, α1B, α1S, β2, or γ. The chosen primers amplified cochlear cDNA to yield an overall pattern of bands different from that of any tissue studied thus far, in particular with respect to the α2δ and β1 subunits; the α2δ product was found to be significantly shorter than the corresponding brain and skeletal muscle isoforms. Nucleotide sequencing confirmed the identity of mouse cochlear subunit cDNAs. The results suggest that L‐type and presumptive R‐type calcium channels are expressed in the mammalian cochlea and that the α2δ subunits may be coded by a characteristic splice‐variant mRNA.

Analysis of γ-aminobutyric acidA receptor subunits in the mouse cochlea by means of the polymerase chain reaction

Abstract: Thirteen GABAA receptor subunits were examined in the cochlea of the CBAJ mouse by PCR analysis. Total RNA was extracted from the auditory organs of 14–18‐day‐old animals, and mRNA was isolated using oligo‐dT cellulose. After reverse transcription, resulting cDNA was amplified by PCR with primers specific for nucleotide sequences representing GABAA subunits. PCR products corresponding to subunits α1‐αB, β1,‐β3, and γ2 were amplified, whereas those representing γ1, γ3, and δ were not amplified above background. These results provide the most direct evidence to date that GABAA receptors composed of the detected subunits are expressed in the mammalian cochlea, lending new support to previous studies implicating GABA as a co‐chlear transmitter. The pronounced expression of α2 and α6 sub‐units suggests type II and “cerebellar‐type” benzodiazepine pharmacology in the cochlea.

Quantitative analysis of dopamine receptor messages in the mouse cochlea

Dopamine receptor isoforms were examined in the cochlea of the CBA(J) mouse by RT-PCR analysis and nucleotide sequencing, utilizing primers specific for known dopamine receptor isoforms. Cochlear cDNA sequences corresponding to dopamine D2(long) and D3 receptors were amplified, whereas those representing D1A, D1B, D2(short), and D4 were not detected. Utilizing quantitative competitive PCR analysis, relative levels of dopamine receptor transcripts were found to be 0.002, 0.014, 0.016, and 1.000 for D2(long) cochlea, D3 cochlea, D3 brain, and D2(long) brain, respectively. In the context of previously published findings, the current work provides key quantitative evidence necessary to establish that dopamine is a neurotransmitter in the auditory inner ear.

Expression of adenylyl cyclase type I in cochlear inner hair cells

Expression of calcium/calmodulin-activated adenylyl cyclase type I (ACI) mRNA has been determined in the cochlea and in an organ-of-Corti subdissected tissue fraction by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Amplification products of predicted size were obtained from the mouse cochlea and rat organ of Corti with nucleotide sequences corresponding to respective ACI brain transcripts. In addition, ACI template was detected in a rat inner hair cell cDNA library by PCR. Immunoreactivity to ACI has been localized within the organ of Corti to the inner hair cell, with diaminobenzidine staining found in both the cell body and in the stereocilia. Evidence, thus, has been obtained that both ACI transcript and protein are expressed in the inner hair cell, the primary mechanosensory receptor cell of the cochlea. We hypothesize that ACI is activated by calcium influx through a calcium/calmodulin interaction and that this adenytyl cyclase isoform may have a role in modulation of receptoneural afferent transmission and/or mechanosensory transduction in the cochlea.

HCN1 and HCN2 Proteins Are Expressed in Cochlear Hair Cells HCN1 CAN FORM A TERNARY COMPLEX WITH PROTOCADHERIN 15 CD3 AND F-ACTIN-BINDING FILAMIN A OR CAN INTERACT WITH HCN2

Background: Protein-protein interaction between HCN1 and tip-link protocadherin 15CD3 suggests a role for HCN1 in hair-cell mechanotransduction. Results: HCN1 and HCN2 form alternate ternary protein complexes with hair-cell stereociliary proteins. Conclusion: Alternate protein-protein interactions reflect separation of binding events for stereociliary proteins implicated in mechanotransduction. Significance: HCN1 is the only channel component identified as binding to a stereociliary tip-link protein. A unique coupling between HCN1 and stereociliary tip-link protein protocadherin 15 has been described for a teleost vestibular hair-cell model and mammalian organ of Corti (OC) (Ramakrishnan, N. A., Drescher, M. J., Barretto, R. L., Beisel, K. W., Hatfield, J. S., and Drescher, D. G. (2009) J. Biol. Chem. 284, 3227–3238). We now show that Ca2+-dependent interaction of the organ of Corti HCN1 and protocadherin 15 CD3 is mediated by amino-terminal sequence specific to HCN1 and is not replicated by analogous specific peptides for HCN2 or HCN4 nor by amino-terminal sequence conserved across HCN isoforms utilized in channel formation. Furthermore, the HCN1-specific peptide binds both phosphatidylinositol (3,4,5)-trisphosphate and phosphatidylinositol (4,5)-bisphosphate but not phosphatidylinositol 4-phosphate. Singly isolated cochlear inner and outer hair cells express HCN1 transcript, and HCN1 and HCN2 protein is immunolocalized to hair-cell stereocilia by both z-stack confocal and pre-embedding EM immunogold microscopy, with stereociliary tip-link and subcuticular plate sites. Quantitative PCR indicates HCN1/HCN2/HCN3/HCN4 = 9:9:1:89 in OC of the wild-type mouse, with HCN4 protein primarily attributable to inner sulcus cells. A mutant form of HCN1 mRNA and protein is expressed in the OC of an HCN1 mutant, corresponding to a full-length sequence with the in-frame deletion of pore-S6 domains, predicted by construct. The mutant transcript of HCN1 is ∼9-fold elevated relative to wild-type levels, possibly representing molecular compensation, with unsubstantial changes in HCN2, HCN3, and HCN4. Immunoprecipitation protocols indicate alternate interactions of full-length proteins; HCN1 can interact with protocadherin 15 CD3 and F-actin-binding filamin A forming a complex that does not include HCN2, or HCN1 can interact with HCN2 forming a complex without protocadherin 15 CD3 but including F-actin-binding fascin-2.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) and its receptor (PAC1-R) are positioned to modulate afferent signaling in the cochlea

Pituitary adenylyl cyclase-activating polypeptide (PACAP), via its specific receptor pituitary adenylyl cyclase-activating polypeptide receptor 1 (PAC1-R), is known to have roles in neuromodulation and neuroprotection associated with glutamatergic and cholinergic neurotransmission, which, respectively, are believed to form the primary basis for afferent and efferent signaling in the organ of Corti. Previously, we identified transcripts for PACAP preprotein and multiple splice variants of its receptor, PAC1-R, in microdissected cochlear subfractions. In the present work, neural localizations of PACAP and PAC1-R within the organ of Corti and spiral ganglion were examined, defining sites of PACAP action. Immunolocalization of PACAP and PAC1-R in the organ of Corti and spiral ganglion was compared with immunolocalization of choline acetyltransferase (ChAT) and synaptophysin as efferent neuronal markers, and glutamate receptor 2/3 (GluR2/3) and neurofilament 200 as afferent neuronal markers, for each of the three cochlear turns. Brightfield microscopy giving morphological detail for individual immunolocalizations was followed by immunofluorescence detection of co-localizations. PACAP was found to be co-localized with ChAT in nerve fibers of the intraganglionic spiral bundle and beneath the inner and outer hair cells within the organ of Corti. Further, evidence was obtained that PACAP is expressed in type I afferent axons leaving the spiral ganglion en route to the auditory nerve, potentially serving as a neuromodulator in axonal terminals. In contrast to the efferent localization of PACAP within the organ of Corti, PAC1-R immunoreactivity was co-localized with afferent dendritic neuronal marker GluR2/3 in nerve fibers passing beneath and lateral to the inner hair cell and in fibers at supranuclear and basal sites on outer hair cells. Given the known association of PACAP with catecholaminergic neurotransmission in sympathoadrenal function, we also re-examined the issue of whether the organ of Corti receives adrenergic innervation. We now demonstrate the existence of nerve fibers within the organ of Corti which are immunoreactive for the adrenergic marker dopamine beta-hydroxylase (DBH). DBH immunoreactivity was particularly prominent in nerve fibers both at the base and near the cuticular plate of outer hair cells of the apical turn, extending to the non-sensory Hensens cell region. Evidence was obtained for limited co-localization of DBH with PAC1-R and PACAP. In the process of this investigation, we obtained evidence that efferent and afferent nerve fibers, in addition to adrenergic nerve fibers, are present at supranuclear sites on outer hair cells and distributed within the non-sensory epithelium of the apical cochlear turn for rat, based upon immunoreactivity for the corresponding neuronal markers. Overall, PACAP is hypothesized to act within the organ of Corti as an efferent neuromodulator of afferent signaling via PAC1-R that is present on type I afferent dendrites, in position to afford protection from excitotoxicity. Additionally, PACAP/PAC1-R may modulate secretion of catecholamines from adrenergic terminals within the organ of Corti.

The role of cellular degeneration in the normal development of (rat) otocyst

From day 12 to 16 of gestation a highly circumscribed zone of cellular degeneration has been observed in the otocyst of more than 50 normal rats. This zone appears to be produced by selective or programmed cell death, and results in the elimination of unneeded cellular elements. That cells in this zone are also selectively destroying subcellular organelles as a first step in specialization is also discussed.

Cloning and characterization of α9 subunits of the nicotinic acetylcholine receptor expressed by saccular hair cells of the rainbow trout (Oncorhynchus mykiss)

Abstract α9/α10 Subunits are thought to constitute the nicotinic acetylcholine receptors mediating cholinergic efferent modulation of vertebrate hair cells. The present report describes the cloning and sequence analysis of subunits of the α9-containing receptor of a hair-cell layer from the saccule of the rainbow trout ( Oncorhynchus mykiss ). A major α9 subunit, termed α9-I, displayed typical features of a nicotinic α subunit, with total coding sequence of 572 amino acids including a 16 amino-acid signal peptide. It possessed an extended cytoplasmic loop between membrane-spanning regions M3 and M4, compared with mammalian homologs. Transcript for α9-I was robustly expressed in the saccular hair cell layer and less prominently in trout olfactory mucosa, spleen, pituitary gland, and liver, as determined by reverse transcription–polymerase chain reaction. α9-I cDNA was not detected in trout brain, skeletal muscle, retina, and kidney. The α9-I nicotinic receptor protein was immunolocalized, with an affinity-purified antibody directed against a trout α9-I epitope, to hair-cell and neural sites in the saccular hair-cell layer. Foci were found at basal and basolateral membrane sites on hair cells as well as on afferent nerve. Receptor clustering was observed in hair cells bordering non-sensory epithelium. Since in higher vertebrates the α9 is reported to associate with another nicotinic subunit, α10, we examined the possibility of expression of additional nicotinic subunits in trout saccular hair cells. Message for another nicotinic subunit, termed α9-II, was found to be expressed in the hair cells, although more difficult to amplify than α9-I. In contrast to α9-I, α9-II was expressed in brain, as well as in olfactory mucosa, less prominently in pituitary gland and liver, but not in spleen, skeletal muscle, retina, or kidney. The cloned α9-II had a total coding sequence of 550 amino acids, which included a 17-amino-acid signal peptide, and an extended M3–M4 loop. A third nicotinic subunit message, termed α9-III, was PCR-amplified from trout olfactory mucosa where it was strongly expressed. However, message for α9-III was not detected in hair cells. Message for α9-III was moderately expressed in trout brain, retina, and pituitary gland but not in trout spleen, skeletal muscle, liver, and kidney. Thus, α9-I and α9-II may together contribute to the formation of the hair-cell nicotinic receptor of teleosts, where no ortholog of α10 appears to exist. The current work is, to our knowledge, the first description of α9 coding sequences directly from a vertebrate hair cell source. Further, the generality of hair cell expression of subunits for the α9-containing nicotinic cholinergic receptor has been extended to fishes, suggesting a similar efferent mechanism across all vertebrate octavolateralis sensory systems.

Bryostatin 1-induced modulation of the acute lymphoblastic leukemia cell line Reh

Summary We have previously reported that the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) induces further differentiation of the human acute lymphoblastic leukemia cell line Reh to a monocytoid B lymphocyte stage. In the present study, we investigated the differentiating capacity of another protein kinase C (PKC) activator, bryostatin 1 (bryo). Reh cells were treated in vitro with TPA, bryo, or interferon-α (IFN-α) for a period of 5 days during which cells were analyzed for changes in growth patterns, morphology, cytochemistry, and surface phenotype. Bryo caused a dose-dependent growth inhibition of Reh cells. Morphologically, the treated cells expressed monocytoid features with development of filopodia and numerous vacuoles indicating phagocytic activity. Bryo induced similar phenotypic changes to TPA, including induction of CD11c, increased expression of CD22 and down-regulation of CD10 and CD19. Enzymatically, bryo, like TPA, induced tartrate-sensitive acid phosphatase expression but failed to induce periodic acid Schiff (PAS) and nonspecific esterase (NSE). Bryo inhibited the TPA action on NSE and CD10. IFN-α showed additive growth inhibitory and phenotypic effects to bryo. Collectively, our findings indicate that bryo is capable of inducing further differentiation of the Reh cells along the B cell lineage similar to those of TPA.

Pituitary Adenylyl Cyclase-Activating Polypeptide (PACAP) and its receptor (PAC1-R) in the cochlea: Evidence for specific transcript expression of PAC1-R splice variants in rat microdissected cochlear subfractions

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a neuropeptide originally isolated from the hypothalamus, named for its high potency in stimulating adenylyl cyclase in pituitary cells. PACAP acts through the specific receptor PAC1-R to modulate the action of neurotransmitters, and additionally, to regulate cell viability via autocrine/intracrine mechanisms. Evidence has now been obtained that PACAP and multiple splice variants of PAC1-R are expressed in the rat cochlea. mRNA for PACAP precursor protein is found by reverse transcription-polymerase chain reaction (RT-PCR) in microdissected cochlear lateral wall, organ of Corti, and spiral ganglion subfractions. A specific pattern of expression of mRNA for PAC1-R splice variants, which mediate the response to PACAP, has been revealed by RT-PCR and cloning for the cochlear subfractions. Transcript for the short form of PAC1-R is found in all three subfractions. Four additional splice variants -- hop1, hop2, hip, and a novel hop1 splice variant -- are expressed in the lateral wall. For the amino terminus splice region of PAC1-R, a new splice variant has been detected in the organ of Corti, representing a deletion of the first 7 of 21 amino acids detected in the PAC1-R very-short sequence. Overall, from message determinations in cochlear subfractions, there are five PAC1-R splice variants in the lateral wall, two in the organ of Corti and one in the spiral ganglion, indicating multiple possible responses to PACAP and/or mechanisms to modulate the response to PACAP in the cochlea. The variety of PAC1-R splice variants expressed may reflect the diversity in cell function between subfractions that is modulated by PACAP. The neuropeptide and its specific receptor have been immunolocalized in the lateral wall, the source of the largest number of cochlear PAC1-R splice variants. The receptor was targeted by primary antibodies which would elicit immunoreactivity for all splice variants of PAC1-R detected with RT-PCR, and evidence has been obtained with Western blot analysis suggesting that PAC1-R is glycosylated in vivo. Within the lateral wall, PACAP and PAC1-R were immunolocalized primarily to the stria vascularis, with immunoreactivity for both neuropeptide and receptor increasing from the basal to apical cochlear turns. Within the stria, PACAP immunoreactivity was localized to the basolateral extensions of marginal cells, while PAC1-R was clearly associated with tight junctions between the marginal cells close to the endolymphatic compartment. In addition, evidence was obtained that PAC1-R was associated with endothelial cells of the capillaries in the stria vascularis. The large number of splice variants expressed, coupled to the specificity in linkage between PAC1-R splice variants and G-protein-coupled second messenger pathways, could provide a mechanism to closely modulate tight junction integrity in the stria vascularis, impacting the endolymphatic potential.