Neurokinin receptor cell lines

A method for producing high level expression of neuropeptide receptors and stable cell lines useful therein. This method involves culturing a cell line containing amplified copies of a cDNA sequence encoding a neuropeptide receptor containing an expression control sequence taken from the .beta.-actin transcriptional control element.

This invention relates to a novel genetically engineered mammalian cell 
line which is capable of high level expression of neurokinin A receptors. 
BACKGROUND OF THE INVENTION 
Our knowledge of the origin of neurotransmitter receptor subtypes has been 
advanced by the identification of several neurotransmitter genes and the 
pharmacological characterization of the gene products, which are generally 
present in nature in very limited quantities. Within the peptide 
neurotransmitter family are the tachykinins. Mammals contain three 
distinct tachykinin neuropeptides, substance P, neurokinin A (also 
designated NKA and known as substance K), and neurokinin B (also known as 
neuromedin K), and it has been suggested that there are multiple 
tachykinin receptor subtypes. Expression of the recombinant receptor 
protein therefore is an important goal in the characterization of the 
neuropeptide receptors and in understanding their function and 
pharmacological significance. 
This invention relates generally to a expression system for obtaining high 
level expression of selected neuropeptide receptors. More specifically, 
the invention discloses the host cells B82 which express high levels of 
neurokinin A receptors as effected by a cDNA encoding the bovine 
neurokinin A receptor, which has been operatively linked to the human 
.beta.-actin transcriptional control element. 
SUMMARY OF THE INVENTION 
The invention describes a new cell line that has been established from 
transfection of the mouse fibroblast cell line B82 with an expression 
plasmid containing the neurokinin A receptor gene. The new cell line is 
capable of expression of high levels of neurokinin A receptors defined by 
the binding of neurokinin A. The selectable marker chosen for the cloning 
of transfected B82 cells in this case was the aminoglycoside 
phosphotransferase (APH) gene.

DETAILED DESCRIPTION OF THE INVENTION 
Definitions 
Several terms are set forth in the invention. Those terms are meant to have 
the following meanings. 
Amplifying cells: Amplifying cells comprises culturing a cell line 
containing a selectable marker with a selective agent that gives rise to a 
population of cells that have an increased level expression of a desired 
protein. Generally, the result of amplification is an increased copy 
number of a plasmid present either extracellularly or one that has 
integrated into the genomic DNA. Selectable markers chosen include the 
genes for adenosine deaminase(ADA), aminoglycoside phosphotransferase 
(neo, G418, APH), dihydrofolate reductase (DFR), 
hygromycin-B-phosphotransferase(HPH), thymidine kinase (TK), and 
xanthine-guanine phosphoribosyltransferase (XGPRT, gpt). 
Biologically active receptors: A function or set of functions performed by 
a receptor in a biological context, that may either be in vivo in an 
organism or in in vitro in a biochemical system. Biologically active 
receptors may be divided into catalytic and effector activities. 
Catalytic activities: Catalytic activities of receptors generally involve 
the activation and/or suppression of cellular factors and pathways through 
the binding of the ligand. 
Complementary DNA or cDNA: A DNA molecule or sequence which has been 
enzymatically synthesized from the sequences present in a mRNA template. 
DNA or Plasmid Construct: A DNA sequence or plasmid containing such 
sequence which may be isolated in whole or partial form which has been 
modified to contain segments of DNA which are combined and juxtaposed in a 
manner which would otherwise not exist in nature. 
Effector activities: Effector activities of receptors include specific 
binding of the biological ligand. Effector activity frequently augments, 
or is essential to a catalytic activity under physiological conditions. 
Expression: Expression is the use of the sequences from the transfection of 
the plasmid or vector that is transcribed into mRNA which then is utilize 
to direct the synthesis of protein in conjunction with the cellular 
machinery of the host organism. 
Genetic Code: The sequence of nucleotides in a gene that determines the 
order of amino acids in a corresponding protein. The genetic code is the 
relationship between the twenty-letter language of amino acids used in 
proteins to the four letter language of the nucleic acids. The genetic 
code is degenerate in that more than one codon can specify the same amino 
acid. However, all codons are unambiguous in that each specifies no more 
than one amino acid or signal termination of translation of the peptide or 
protein. 
Hybridizing: The process of forming a double-stranded molecule by 
complementary base-paring between two single-stranded DNA molecules, or a 
single-stranded DNA molecule and an RNA molecule. As such plasmid, 
vectors, or portions thereof, or DNA derived from pieces of synthetic 
synthesized regions can be used to detect a complementary base-pairing 
sequence that has a complementary or homologous sequence, often referred 
to as a probe. The probe is invariably labeled with a radioactive, 
fluoresing, or biotin derivative so as to allow autoradiographic or 
enzymatic detection of the hybridization reaction. 
Joined: DNA sequences are said to be joined when the 5' and 3' ends of one 
DNA sequence are attached, by phosphodiester bonds, to the 3' and 5' ends, 
respectively, of an adjacent DNA sequence. Joining may be achieved by such 
methods as enzymatic ligation of blunt or cohesive termini, by synthesis 
of joined sequences through cDNA cloning, or by deletion, insertion, or 
change of internal sequences by a process of directed mutagenesis. 
Mutagenesis: The process whereby DNA sequences are altered. Such changes in 
the DNA sequence may be employed to bring about an alteration in the 
functional activity of the sequence or affect the nature of protein 
sequence that may be encoded within the sequence. Amongst the numerous 
techniques employed to bring about changes in DNA sequences are the use of 
synthetic oligonucleotides to direct the desired change. Such changes may 
insert, delete, or change the surrounding nucleotide sequences. One may 
also change DNA sequences using chemical mutagens or by placing the cells 
in which the DNA resides under selective environmental pressures so as to 
effect a change in the DNA. 
Non-biologically active receptors: Receptors that may be defective in 
either their catalytic or effector activities, or both. Receptors of this 
type would be able to be produced through mutagenesis of the receptor 
sequence. 
Operative association: A transcriptional control sequence is in operative 
association when when a promoter and its associated regulatory elements 
are so joined to a gene that it operates to transcribe said gene. 
Plasmid or Vector: A DNA construct containing genetic information which may 
provide for its replication when inserted into a host cell. A plasmid 
generally contains at least one gene sequence to be expressed in the host 
cell, as well as sequences which facilitate such expression, including 
promoters and transcription initiation sites. It may be a linear or a 
closed circular molecule. 
Selection: Any means of identifying a clone that contains a desired 
recombinant DNA molecule. Such means of identification may include a 
selectable marker whereby the gene carried by a vector that confers a 
recognizable characteristic to a cell containing the vector. 
Transcriptional control sequences: Those DNA sequence elements that provide 
for transcription and termination of aligned genes by RNA polymerase II. 
The DNA sequences involved for RNA polymerase II recognition, initiation, 
and termination are not meant as a limit upon the presence of other 
sequences. The transcription control sequence may also include sequences 
and associated transcription factors that would enable an operational 
transcription unit. Addition of enhancers, tissue specific regulatory 
elements, and maintence factors all fall within the scope of 
transcriptional control sequences. Furthermore, such sequences need not be 
juxtaposed to the transcription initiation and termination sequences and 
thereby may be positioned 5' or 3' to these sequences, including those 
inserted in intervening sequences and serves therein as a part of the 
transcriptional unit. 
Transfection: Is the introduction of DNA into cultured cells generally for 
the purpose of acquiring new genes in a cell. 
Description and Maintenance of Cell Lines: 
The B82 cell line is a mouse L cell line which is a heteroploid 
fibroblast-like cell that is thymidine kinase deficient and is selectable 
with 5-bromodeoxyuridine. 
Construction of Neurokinin A Receptor Expression Vectors 
Clones for the neurokinin A receptor were isolated from a cDNA library from 
bovine stomach mRNA (Masu et. al., Nature 329, 836-838). The bovine cDNA 
library was constructed in a lamda expression vector containing the SP6 
and T7 promoters to provide a mechanism to transcribe inserted 
heterologous genes in vitro. mRNAs were expressed in functional form 
following microinjection of the mRNA in frog oocytes, previously shown to 
have the cellular machinery to express the exogenous receptor mRNA. A cDNA 
clone for the bovine neurokinin A (NKA) receptor was isolated using 
electrophysiological measurements of the injected Xenopus oocytes. 
Individual clones expressing neurokinin A receptors were able to be found 
by electrophysiological changes of the oocytes capable of responding to 
neurokinin A through the NKA receptor. 
Several NKA receptor cDNA clones were found in this manner and their 
structures determined. The clone pSKR56S was chosen for expression in the 
instant invention. The cDNA insert of this clone was initially subcloned 
into the cloning vector pGEM3 and was used as the source of DNA for 
cloning into the expression vector pH.beta.APr-1-neo. All reconstructions 
of the vector and subcloning were done in E. Coli strains and are within 
the scope of someone skilled in the art. 
Expression of the bovine neurokinin A gene utilized the elements present in 
the expression vector pH.beta.APr-1-neo (Gunning, et al., PNAS 
84,4831-4835). The essential elements of the vector consist of the 5' 
flanking sequence of the human .beta.-actin promoter and the first 
intervening sequence (IVS1) linked to unique restriction sites for 
insertion of exogenous genes, and a simian virus 40 (SV40) polyadenylation 
signal. The restriction sites 3' to the promoter for insertion of 
exogenous genes are derived from the multiple cloning region of pSP64. The 
expression elements from SV40 of pH.beta.APr-1-neo were derived from the 
framework of the Okayama-Berg expression vector pcDV1 and used in the 
creation of the parent vector pH.beta.APr-1. pH.beta.APr-1-neo was a 
subsequent modification of pH.beta.APr-1 to incorporate the 
neomycin-resistance gene (directed by the SV40 early promoter) to allow 
for selection of cells that stably integrate exogenous DNA. The neomycin 
gene and SV40 promoter were taken from the plasmid pSV2neo as a EcoRI/ 
PvuII DNA fragment and inserted between the EcoRI and ClaI sites of 
pH.beta.APr-1 to create pH.beta.APr-1-neo. 
pH.beta.APr-1-neo-SKR is the instant vector for expression of the 
neurokinin A receptor. Cloning of the NKA receptor gene into 
pH.beta.APr-1-neo was accomplished by moving the HindIII-BamHI fragment in 
pGEM3, which includes the NKA receptor gene derived from pSKR56S, and 
joining it to the parent expression vector pH.beta.APr-1-neo. 
Vector and DNA 
The procedures for plasmid construction and DNA preparation are well known 
in the art and have been produced by a number of authorities (Maniantis, 
et al, Molecular Cloning (1982); Ausubel, et al. Current Protocols in 
Molecular Biology). All plasmids were grown in the Escherichia coli host 
HB101 or TG1. The plasmids have all been described. 
The vectors for use in producing the cells or cell lines useful in the 
method of the present invention are preferably supercoiled, 
double-stranded circular constructs. All enzymes are commercially 
available and are utilized using procedures well known to those trained in 
the art, and are well described in the literature and by the commercial 
suppliers. Nucleotide sequencing was performed using the dideoxy 
chain-termination method or can be performed by other well known standard 
techniques in the art. 
Transfection of Mammalian Cell Lines 
Generally three techniques are currently used for introduction of DNA into 
mammalian cells: calcium phosphate transfection, DEAE-dextran 
transfection, and electroporation. The first two procedures produce a 
chemical environment that results in the DNA attaching to the cell 
surface. The DNA is then endocytosed into the cell. Electroporation uses 
an electric field to create pores in the cell membrane, and the presumably 
the DNA enters through the pores. Generally, it is important to optimize 
the protocol for a given cell type. The concentration of DNA to be 
transfected, the concentration of the active agent for the transfection, 
and the time course of expression and selection of the transfected gene 
are all variables that must be determined for use and are within the skill 
of a person trained in the art. 
Selection and Amplification of Recombinant Cells 
Approximately one in 10.sup.4 cells in a transfection will stably integrate 
DNA, so a dominant selectable marker is used to permit isolation of stable 
transfectants. Appropriate selection conditions for the parental cell line 
are first determined and then applied to the transfected cells and allowed 
to grow under selection for approximately 10 doublings before individual 
colonies are chosen and expanded into cell lines. Several markers are 
commonly used for selection of mammalian cells. The selectable markers 
include adenosine deaminase(ADA), aminoglycoside phosphotransferase(neo, 
G418, APH), dihydrofolate reductase (DFR), 
hygromycin-B-phosptransferase(HPH), thymidine kinase (TK), and 
xanthine-guanine phosphoribosyltransferase (XGPRT, gpt). 
Upon transfection with pH.beta.APr-1-neo-SKR, the cell population is 
exposed to environmental pressure (G418) sufficient to require the cell to 
produce more copies of the amplifiable gene, e.g. APH (aminoglycoside 
phosphotransferase), for survival. The cells are examined for the presence 
of the marker and those cells which have successfully incorporated the 
vector DNA with the marker gene will exhibit the marker phenotype. Thus 
when a cell is transfected with DNA containing the exogenous gene and a 
selectable marker, this enables the identification of those cells which 
have incorporated the DNA by selection of those cells that are able to 
survive drug treatment by expressing the requisite marker. The exogenous 
gene and the marker gene may be contained on a contiguous stretch of DNA 
or co-transfected on independent and separate stretches of DNA. 
G418 is an antibiotic that blocks protein synthesis in mammalian cells by 
interfering with ribosomal function and is the basis for selection in the 
instant invention. G418 is an aminoglycoside, similar in structure to 
neomycin, gentamycin, and kanamycin. Expression of the bacterial APH gene 
(derived from the transposon tn5) in mammalian cells allows recombinant 
cells to survive by their ability to detoxify G418. (Southern, et. al., J. 
Mol. Appl. Gen. 1:327-341 (1982)). 
Once the host cell line is transformed with the vector with the selectable 
gene and the gene coding for neurokinin A receptors, and the desired 
transformants are selected, they are screened for integration of the NKA 
receptor gene into the host chromosome or for expression of the receptor 
protein itself. Screening for expression of the integrated receptor gene 
can utilize standard immunological or biological assays. Once the 
transformants have been identified, expression of the receptor gene can be 
again amplified by subculturing the cells in the presence of the selecting 
agent, in constant or increasing concentrations, to yield cells producing 
higher levels of the receptor protein (1-500 femtomoles receptor per mg 
total protein in the assay). It should be noted that in the practice of 
this invention, transcription units may in general contain either cDNA or 
genomic DNA encoding neurokinin A receptors, a general marker such as 
neomycin resistance gene, and general transcriptional control sequences in 
operative association therewith. 
Cells expressing neurokinin A receptors can be determined utilizing a 
radiolabeled-ligand binding assay. Essentially cell membranes from the 
cells to be determined are prepared from a cell lysate and a differential 
centrifugation of the membranes. The cell-membrane pellet is subsequently 
used in the binding assay. The binding assay consists of incubating the 
cell membranes in the presence of a radiolabeled ligand and determining 
the adsorption of the ligand into the membranes. Free versus bound ligand 
is generally separated by use of a filter binding assay. Specific binding 
is defined as the difference in binding between tubes containing no test 
compound and tubes containing unlabeled neurokinin A. Total membrane bound 
radioactivity is generally approximately 5% of that added to the tubes. 
Specific binding is generally 85-95% of total binding. 
Biologically Active Receptors 
One of the features of biologically active neurokinin A receptors includes 
the activation of a G protein, which inturn activates phospholipase C 
(PLC). PLC converts to phosphatidylinositol phosphate (PI) into inositol 
1,4,5-triphosphate (IP.sub.3) and diacyglycerol (DAG). IP.sub.3 and DAG 
are believed to be intracellular signals of neurokinin A receptor 
activation. 
EXPERIMENTAL PROCEDURE: 
EXAMPLE 1 
Construction of Cell Lines 
B82 and transfected B82 cells are maintained in a medium containing 45% 
Dulbecco's modified Eagles's medium, 45% Ham's F-12 medium, 5% fetal 
bovine serum, 5% newborn calf serum, 100 U/ml of penicillin and 100 ug/ml 
of streptomycin. Cultures of transfected cells were periodically 
supplemented with 300 ug/ml of the selective antibiotic geneticin (G418; 
Gibco, Grand Island, N.Y.); . Cells were grown in Costar 75 cm.sup.2 
tissue culture flasks and incubated in a humidified atmosphere of 95% air 
and 5% CO.sub.2. 
Example 2 
Introduction of the Neurokinin A Receptor cDNA into Mammalian Expression 
Vectors 
The bovine stomach cDNA clone, pSKR56S, was purified from a culture of its 
E. coli bacterial host utilizing standard alkaline lysis procedures. 
Several other suitable procedures for purifying plasmids are known to 
those skilled in the art. 
The bovine cDNA in the plasmid was excised with suitable restriction 
endonucleases and ligated to the eukaryotic expression vector, 
pH.beta.APr-1-neo. For the construction of the expression plasmid 
pH.beta.APr-1-neo-SKR, the bovine cDNA in pSKR56S was first excised with 
the endonucleases XbaI and BamHI and ligated to the cloning vector pGEM3. 
This recombinant DNA was cloned and amplified, followed by HindIII and 
BamHI digestion to recover the cDNA insert, now flanked by a 5' HindIII 
end and a 3' BamHI end. These restriction sites facilitated the 
directional cloning of the bovine CDNA into the expression vector, 
pH.beta.APr-1-neo, which has a unique HindIII (5') and BamHI (3') site 
downstream from the promoter element for insertion and expression of 
heterologous genes. Cloning of the cDNA into the vector was accomplished 
using E. coli as the host and then purified from the host as a pure 
plasmid DNA preparation. 
Example 3 
Transfection of Mammalian Cells 
The plasmid pH.beta.APr-1-neo-SKR, was transfected into the mammalian 
fibroblast L cell line, B82, by the calcium phosphate precipitation 
method. (Wigler et al., Cell 14, 727 (1978)). Essentially a precipitate 
containing CaPO.sub.4 and DNA is formed by slowly mixing a HEPES-buffered 
saline solution with a solution containing CaCl.sub.2 and DNA. The 
HEPES-buffered saline (HBS) was made up as a 10x concentrate containing 
8.18% NaCl (w/v), 5.94% Hepes (w/v), 0.2% Na.sub.2 HPO.sub.4 (w/v). For 
transfection the 10x solution was diluted to 2x with dH.sub.2 O and the pH 
adjusted to 7.12 with 1N NaOH. This solution was filter sterilized before 
use. To prepare the DNA precipitate, 10-20 .mu.g of supercoiled plasmid 
DNA was mixed with 31 .mu.l of 2M CaCl.sub.2 and brought to a final volume 
of 250 .mu.l with sterile dH.sub.2 O. This mixture was then added dropwise 
to an equal volume of 2x HBS. This mixture (500 .mu.l) was added to the 
culture medium of a 25 cm.sup.2 flask which had been seeded with 10.sup.4 
cells/cm.sup.2 of B82 cells the day before transfection. The cells were 
immediately returned to the incubator and incubated for 3.5-4 hours. At 
the end of this incubation, a glycerol shock was usually carried out using 
0.5 ml of 15% glycerol/HBS per flask for 1 min at 37.degree. C. The cells 
were then rinsed with phosphate buffered saline and returned to the 
incubation medium as described above in a humidified atmosphere and 5% 
CO.sub.2 for 48 hours prior to the addition of G-418 at 500 ug/ ml of 
medium. At this point the cells from each flask were transferred to two 
100 mm petri dishes and the culture medium replaced with fresh medium that 
had been supplemented with 500 .mu.g/ml of G-418. The medium was 
replenished after 4 days, and the cells were cultured in this supplemented 
medium until visible colonies formed. These colonies represent clonal 
cells which are resistant to G-418. The colonies, typically 1-3 mm in 
diameter, were harvested at random and expanded for subsequent screening 
procedures. 
Example 4 
Selection and Amplification of Integrated Vector 
Neomycin resistant B82 colonies or clones were assayed for the presence of 
the protein product which was encoded by the foreign gene transfected into 
these cells. In the case of the instant clones, the bovine cDNA 
transfected into the B82 cells had previously been shown to encode for 
neurokinin A receptors. 
The expression of the neurokinin A receptors in the B82 clones, categorized 
by the systematic name of SKLKB82#n (n of the systematic name designates 
different clonal isolates of the transfected cells). SKLKB82#n clones are 
stable with passage and culturing conditions for these cells are the same 
as that of the wild type B82 cells. The cell line SKLKB82#3 has been 
deposited with ATCC (ATCC CRL 10320). 
The presence of the receptor protein in the B82 clones was established by a 
series of radiolabeled neurokinin A receptor binding assays. Several 
clones had observable levels of NKA receptors indicating that the bovine 
cDNA had been integrated into the genome of the B82 cells and was 
biologically and functionally being expressed. 
Example 5 
Characterization of Functional Neurokinin A Receptor in Trasfected B82 
Cells by Receptor Binding 
Pellets of scraped cells of SKLKB82#n are homogenized with a Polytron 
(setting #6 for 15-30 sec) in 15 volumes of 50 mM TRIS-HCl buffer (pH 7.4, 
4.degree. C.) containing 120 mM NaCl and 5 mM KC1. The homogenate is 
centrifuged at 48,000.times.g for 10 min at 4.degree. C. The pellet is 
resuspended (Polytron as above) in 15 volumes of 50 mM TRIS-HCl buffer (pH 
7.4, 4.degree. C.) containing 10 mM EDTA and 300 mM KC1and allowed to set 
on ice for 30 min. The suspension is centrifuged as above and the pellet 
is washed by resuspension and centrifugation two additional times in plain 
50 mM TRIS-HCl buffer (pH 7.4, 4.degree. C.). The final pellet is 
resuspended in a concentration of 1-10 mg/ml in incubation buffer (see 
below) and allowed to set at room temperature for approximately 15 minutes 
before use. The binding assay (Burcher, E., et al., J. Pharmacol. Exp. 
Ther., 236:819-831, 1986.) is carried out in duplicate or triplicate in 12 
mm.times.75 mm polypropylene culture tubes in incubation buffer consisting 
of 50 mM TRIS-HCl (pH 7.4, room temperature) containing 0.1% bovine serum 
albumin, 2 mM MnCl.sub.2, 40 ug/ml bacitracin, 4 ug/ml leupeptin, 4 ug/ml 
chymostatin, and 1 uM thiorphan. 0.25 nM ligand (400,000-500,000 CPM) 
diluted in incubation buffer is added to each tube in 50 ul. Test compound 
(10-.sup.10 M to 10-.sup.5 M) diluted in incubation buffer is added to 
appropriate tubes in 50 ul. The assay is started by the addition 250 ul (5 
mg tissue) of well-mixed membrane suspension. The final incubation volume 
is 0.5 ml. The assay is carried out at room temperature for 2 hr. The 
incubation is quickly terminated by the addition of 3.5 ml of plain 
TRIS-HCl buffer (pH 7.4, 4.degree. C.) and filtration under vacuum through 
GF/B filters that have been presoaked overnight in 0.5% BSA. The filters 
are rapidly washed with two 3.5 ml portions of the same buffer and 
transferred to 12 mm.times.75 mm polystyrene culture tubes for direct 
counting in a gamma scintillation counter. Specific binding is defined as 
the difference in binding between tubes containing no test compound and 
tubes containing 1 uM unlabeled neurokinin A. Total membrane bound 
radioactivity is generally approximately 5% of that added to the tubes. 
Specific binding is generally 85-95% of total binding. 
Protein content of the crude membrane suspension can be determined (Lowry 
et al., J. Biol. Chem., 193:265, 1951) on an aliquot taken during the last 
tissue wash resuspension (plain 50 mM TRIS-HCl buffer). 
Receptor binding experiments were utilized to identify and characterize B82 
cells transfected with the Neurokenin A receptor expression plasmid 
pH.beta.Apr-1-neo-SKR. Results of these experiments are present in FIGS. 
1-4. 
INTERPRETATION OF RESULTS 
IC.sub.50 is the molar concentration of compound that causes 50% inhibition 
of ligand binding(=K.sub.1 is this particular assay; expressed as 
cumulative mean .+-.S.E.M.! of means from N separate experiments). 
Thereby from a Scatchard analysis of the data, the Bmax is obtained from 
the X-intercept and K.sub.D is obtained from the negative inverse slope of 
a Scatchard plot. 
Example 6 
.sup.3 H-Inositol-1-phosphate accumulation 
The quantification of IP.sub.1 was modified from Berridge et al. SKLKB82#3, 
a transfected cell line with high specific binding, was seeded into 1.6 cm 
diameter culture wells at 125,000/well. After a twenty four hour 
incubation at 37.degree. C. under 95% humidified air/5% carbon dioxide, 
the media was removed and 0.5 ml Iscove's modified Dulbecco's medium 
(IMDM, Irvine Scientific) with 0.2 .mu.M myo-2,3H!inositol (20 Ci/mmol, 
NEN, Boston, Mass.) was added to the well. The culture was incubated for 
22-24 hours under the same conditions. After this incubation, the cells 
were washed once with 1.0 ml of IMDM for ten minutes and incubated for ten 
minutes in 0.2 ml of incubation buffer (IMDM with 10 mM LiCl, 40 ug/ml 
bacitracin, 4 ug/ml leupeptin, and 4 ug/ml chymostatin). The agonists, 
NKA, SP (Peninsula Laboratories, Belmont, Calif.), and senktide (Bachem, 
Philadelphia, Pa.) were individually diluted in the incubation buffer 
before addition to the wells. For the antagonist assay, the incubation 
buffer also contained 10 uM L-659,877 (Merck Neurokinin A antagonist; A. 
T. McKnight, Regulatory Pep. 22, 127, 1988). The reaction was terminated 
in 60 minutes by aspiring the media and adding 0.5 ml methanol. The cells 
were scraped from the wells and placed into chloroform resistant tubes. A 
second aliquot of 0.5 ml methanol was used to collect the remaining cells. 
The cell suspensions were mixed with 1.0 ml chloroform and 0.5 ml double 
distilled water to obtain a two phase separation. After centrifugation at 
4200g for 10 minutes, 0.9 ml aliquots of the upper phase were added to 2.0 
ml of distilled water and mixed. This mixture was passed through poly prep 
columns (Bio-Rad Laboratories, Richmond, Calif.) which consisted of 2.0 ml 
slurry of ten percent anion exchange resin in formate form (AG1-X8, 
100-200 mesh, Bio-Rad Laboratories, Richmond, Calif.). The columns were 
washed three times with 5.0 ml of distilled water and two times with 5.0 
ml of 5 mM sodium tetraborate/60 mM sodium formate (Baker, Phillipsburg, 
N.J.). 3H!IP1 was eluted with 2.0 ml of 0.2M ammonium formate/0.1M formic 
acid (Mallinkrodt, St. Louis, Mo.). The eluate was mixed with nine ml of 
Aquamix (West Chem., San Diego, Calif.) and the radioactivity was counted. 
.sup.3 H-Inositol-1-phosphate accumulation experiments were performed on 
B82 cells tranfected with the Neurokinin A receptor expression plasmid 
pH.beta.Apr-1-neo-SKR. Results of this experiment are present in FIG. 5.