Evidence for LHRH-receptor expression in human airway epithelial (Calu-3) cells and its role in the transport of an LHRH agonist.
OBJECTIVE
To determine whether LHRH-receptor is expressed in Calu-3, a human bronchial epithelial cell line, and to further determine whether this receptor plays a role in the transport of deslorelin, an LHRH agonist.


METHODS
Using cultured monolayers of Calu-3 grown at air-interface, the presence and localization of LHRH-receptors in Calu-3 cells was determined using immunochemical methods. To determine the mechanisms of deslorelin transport, the directionality [apical-basolateral (A-B) and basolateral-apical (B-A)] of deslorelin transport across Calu-3 monolayers and the effects of temperature (37 degrees C and 4 degrees C) and an energy depletor (2,4-dinitrophenol) were investigated. To determine the role of LHRH-receptor in deslorelin transport across Calu-3 monolayers, the influence of an LHRH-receptor antisense oligonucleotide on the LHRH-receptor expression and deslorelin transport was studied. Also, the effect of a competing LHRH agonist, buserelin, on deslorelin transport was determined.


RESULTS
Immunofluorescence studies indicated the predominance of LHRH-receptor in Calu-3 cells at the apical and lateral surfaces. Western blot and RT-PCR studies further confirmed the expression of LHRH-receptor in Calu-3 cells. Deslorelin transport across Calu-3 monolayers was vectorial, with the cumulative A-B transport (1.79 +/- 0.29%) at the end of 240 min being higher than the B-A transport (0.34 +/- 0.11%). Low temperature as well as 2,4-dinitrophenol abolished this directionality. LHRH-receptor antisense oligonucleotide decreased the receptor expression at the mRNA and protein level and reduced the A-B deslorelin transport by 55 +/- 4%, without affecting the B-A transport, suggesting a role for LHRH-receptor in the vectorial transport of deslorelin. In addition, buserelin reduced the A-B deslorelin transport by 56 +/- 5% without affecting the B-A transport.


CONCLUSIONS
Taken together, our results provide evidence that deslorelin is transported across the respiratory epithelium via the LHRH-receptor.
Koushik
Kavitha
K
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
Bandi
Nagesh
N
Sundaram
Sneha
S
Kompella
Uday B
UB
D019297:2,4-Dinitrophenol / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western / Q000379:methods; D002064:Buserelin / Q000493:pharmacokinetics* / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D017097:Electric Impedance; D004847:Epithelial Cells / Q000166:cytology* / Q000502:physiology; D005455:Fluorescent Antibody Technique / Q000379:methods; D015870:Gene Expression / Q000235:genetics*; D006801:Humans; D020320:Oligoribonucleotides, Antisense / Q000494:pharmacology; D012333:RNA, Messenger; D051381:Rats; D011966:Receptors, LHRH / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000276:immunology; D012137:Respiratory System / Q000166:cytology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature; D013997:Time Factors; D017329:Triptorelin Pamoate / Q000031:analogs & derivatives* / Q000493:pharmacokinetics* / Q000494:pharmacology
D020320:Oligoribonucleotides, Antisense; D012333:RNA, Messenger; D011966:Receptors, LHRH; D017329:Triptorelin Pamoate; D002064:Buserelin; D019297:2,4-Dinitrophenol; C066438:deslorelin