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`
`R E V I EW
`
`Calcitonin Gene-Related Peptide and
`Regulation of Human Cardiovascular
`Homeostasis
`
`Jacek J. Preibisz
`
`Isolation of calcitonin m R NA initiated studies on
`the multigene complex encoding a family of pep(cid:173)
`tides: calcitonin, its terminal flanking peptides, cal(cid:173)
`citonin gene-related peptide (CGRP), and amylin.
`C G RP is expressed in a- and /?-forms that vary by
`one and three amino acids in rat and h u m a n s, re(cid:173)
`spectively. Both a- and /?-CGRP are very similar in
`their biologic activities, therefore the role of dupli(cid:173)
`cating the c a l c i t o n i n / C G RP gene is unclear. C G RP
`behaves principally as a regulatory neuropeptide
`acting locally through interaction with target organ
`receptors that are either cyclic-AMP dependent, or
`capable of activating KA TP channels of vascular
`smooth muscle. T he dense distribution of C G R P-
`rich structures and the expression of m R NA in the
`central nervous system suggests that C G RP has a
`neuromodulator or neurotransmitter role not lim(cid:173)
`ited to vasoregulatory effects only, but like calci(cid:173)
`tonin, extends its action to physiologic, metabolic,
`and behavioral functions. Activation of perivascu(cid:173)
`lar sensory nerves stimulates the release of n e u r o(cid:173)
`peptides, including C G R P, w h i ch exerts a potent
`
`The recent isolation and characterization of cal(cid:173)
`
`citonin m R NA initiated studies on the multi-
`gene complex encoding a family of peptides:
`calcitonin, its terminal flanking peptides, calci(cid:173)
`tonin gene-related peptide
`(CGRP),
`and
`amylin
`
`Received from the Department of Medicine and the Hypertension
`Center, The New York Hospital—Cornell Medical Center, New York,
`New York.
`Address correspondence and reprint requests to Dr. Jacek J. Prei(cid:173)
`bisz, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110.
`
`vasodilatory effect on venous and arterial vascula(cid:173)
`ture. T he increased levels of CGRP-like i m m u n o r e(cid:173)
`activity were observed in v o l u me overload states,
`in heart failure and myocardial infarction, and in
`some forms of hypertension. T he beneficial effect
`of C G RP infusions was demonstrated in patients
`with congestive heart failure and also in subjects
`with neurological deficits after surgical treatment
`of subarachnoid hemorrhage. On the other hand,
`there are experimental studies on the inhibition of
`increased C G RP activity, in septic and shock condi(cid:173)
`tions, in w h i ch the vascular hyperrelaxation could
`h a ve deleterious effects. In s u m m a r y, the calcitonin
`gene-related peptide is an attractive and potent
`substance involved in h u m an physiopathology, but
`its role is still not well understood and merits fur(cid:173)
`ther extensive investigation. Am J Hypertens
`1 9 9 3 ; 6 : 4 3 4 - 4 50
`
`KEY W O R D S: Calcitonin gene-related peptide,
`CGRP-like immunoreactivity, vasodilation, volume
`overload, hypertension.
`
`(CGRP-like diabetes-associated p e p t i d e ) .1 - 11 Calci(cid:173)
`tonin, which is synthesized in thyroid C-cells, has been
`historically recognized as one of the calcium regulating
`hormones involved in bone mineral homeostasis.12 In
`contrast to CGRP, the potent vasodilating peptide, the
`role of calcitonin in cardiovascular homeostasis is lim(cid:173)
`ited, both under normal conditions and in the presence
`of the excessively large amounts of this peptide pro(cid:173)
`duced in medullary thyroid c a r c i n o m a .1 2"17 Calcitonin
`lowers the plasma calcium level, modulates the intracel(cid:173)
`lular free calcium influx, and inhibits osteoclastic bone
`
`1
`
`EX2149
`Eli Lilly & Co. v. Teva Pharms. Int'l GMBH
`IPR2018-01426
`
`

`

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`
`Λ/Η-MAY 1993-VOL 6, NO. 5, PART 1
`
`CGRP AND CARDIOVASCULAR HOMEOSTASIS
`
`435
`
`resorption. C G R P, which has a 3 0% homology with
`calcitonin is about 1000-fold less potent in
`lowering
`plasma calcium level and inhibiting the osteoclastic ac­
`tivity, and its effects are most likely exerted via the calci­
`tonin r e c e p t o r .1 8 - 21 The mode of action of calcitonin,
`and in part also of C G R P, involves second messengers
`interacting with the effector system via the guanine nu­
`cleotide binding proteins (G-proteins). Cyclic A MP is a
`well recognized second messenger responsible for slow­
`ing down and cessation of cellular motile p r o c e s s e s .2 1 , 22
`The other postulated second messenger is calcium itself,
`which modulates reduction of cell surface area. Calci-
`tonin-induced inhibition of osteoclastic activity (bone
`resorption) markedly diminishes the calcium flow from
`bones to blood thus lowering the plasma calcium levels.
`Calcitonin
`is also
`involved
`in mineral homeostasis
`through the effect on kidneys, mediated probably by
`prostaglandin E2, to enhance the production of 1,25-di-
`hydroxy-vitamin D .2 3 , 24 The precise role of calcitonin in
`bone mineralization is not well known, but some au­
`thors suggest that the main action of this peptide is to
`preserve bone mass during periods of calcium stress,
`such as Paget's bone disease or pregnancy and lacta­
`tion.2 4 , 25 Elevation of C G RP blood levels throughout the
`gestation period suggests that the increased activity of
`this potent vasoactive peptide could have some effect, in
`contrast to calcitonin, on vascular rather than mineral
`homeostasis during pregnancy.25 Among its other ac­
`tions, calcitonin relieves pain in advanced malignancy
`by affecting serotonergic midbrain nuclei, opiate recep­
`tors, and the prostaglandin-thromboxane s y s t e m .1 2 , 24
`Calcitonin also stimulates gastric acid secretion and in­
`testinal motility, as well as inhibiting the release of sev­
`eral hormones, such as prolactin, growth hormone,
`thyrotropin, and thyrotropin-releasing h o r m o n e .2 4 , 2 6"28
`CGRP, which structurally is very similar to calcitonin
`but mimics only some of its activities, has been recog­
`nized as an important peptide involved in neuromodula-
`tion and in regulation of blood f l o w .1 , 5 , 2 9"33 The struc­
`ture of calcitonin/CGRP gene and its most important
`sequences will be discussed briefly in this review. H o w­
`ever, most of the emphasis will be focused on the distri­
`bution and regulation of C G RP release, its tissue and
`blood levels, and on the effects and behavior of C G RP in
`relation to h u m an vascular homeostasis.
`
`C A L C I T O N I N / C G RP G E NE A ND m R NA
`E X P R E S S I ON
`
`In 1 9 8 1, Rosenfeld et al,9 working with serially trans­
`planted rat cells from medullary thyroid carcinoma, no­
`ticed that these cells had a spontaneous ability to switch
`from high to low calcitonin producing state by increas­
`ing the size of m R N A. Cloning the altered m R NA en­
`abled researchers to locate its sequence on a map of the
`calcitonin gene as well as predict and isolate from rats a
`37-amino acid peptide with amino and carboxyl termi­
`nals named calcitonin gene-related peptide (CGRP) (Fig­
`
`ure 1). In 1984, based on the predicted sequence of rat
`C G R P, a n ew peptide with a similar structure was iso­
`lated from the h u m an medullary thyroid carcinoma.1 ,7
`Using cell-free protein synthesis systems and specific
`antibodies, it was shown that the calcitonin's m R NA is
`— 1000 bases long and in humans encodes a precursor
`protein of 142 amino acids (136 in rats). The procalci-
`tonin's precursor contains calcitonin (32 amino acids)
`itself and the amino and carboxyl terminal
`flanking
`peptides. These peptides are separated from calcitonin
`by a pair of amino acids at the amino terminal and by
`four amino acids at the carboxyl terminal. The matura­
`tion of calcitonin or C G RP prohormones results in exci­
`sion of terminal flanking peptides like calcitonin car-
`boxyl-terminal peptides (CCPI and CCPII) and P D N - 21
`( k a t a c a l c i n ) .3 1 , 3 4 , 35 These peptides vary considerably
`among animal species, some of them exhibiting close
`homology to the h u m an amino acid sequence, but their
`role as regulatory peptides is not yet recognized. CCPII
`and P D N - 21 have been found in the plasma and thyroid
`tissue of medullary thyroid patients, implying that the
`active translation of these novel m R N As could be used
`as a tumor m a r k e r .1 4 , 34 The regulatory pattern of the
`calcitonin gene alternative splicing is not well under­
`stood. Several models have been proposed to explain
`the sequential nature of alternative splicing in different
`genes located on chromosome 1 1, but none were able to
`detect any modulators of p r e - m R NA secondary struc­
`tures. The diversity of calcitonin gene regulatory process
`is
`furthermore demonstrated by gene duplication.
`H u m an C G RP obtained from medullary thyroid carci­
`n o ma (MTC) was first isolated and sequenced as an
`α-form (Figure 2 ) .7 The second gene, also located on
`chromosome 1 1, called /?-calcitonin/CGRP gene, en­
`codes the /J-CGRP sequence, expressed both in humans
`and r a t s .3 6"41 /7-CGRP varies from the α-form by one
`and three amino acids in rat and humans, respectively.
`Both a- and /?-CGRP are very similar in their biologic
`activities, therefore the role of duplicating the C G RP
`gene is unclear. O ne compelling hypothesis claims that
`duplication provides the way to produce C G RP inde­
`pendently of calcitonin, which would be important at
`sites or in circumstances where the inadvertent produc­
`tion of calcitonin could be deleterious.24 The m R NA
`expression of h u m an /?-CGRP has been shown to be less
`than one-tenth that of α - C G R P ,37 nevertheless, plasma
`and cerebrospinal
`fluid
`(CSF) have roughly equal
`amounts of a- and /?-forms.4 Immunochemical methods
`and specific radioreceptor assays are not
`sensitive
`enough to identify conclusively these two peptides.
`
`C G RP R E C E P T O RS A ND T HE R E G U L A T I ON
`OF S E C R E T I ON
`
`Calcitonin gene-related peptide behaves principally as a
`regulatory neuropeptide acting locally through interac­
`tion with target organ receptors that are either cyclic
`A MP dependent, such as calcitonin receptors, or capa-
`
`2
`
`

`

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`
`Ν - terminal
`peptide
`
`ι
`
`Cafcrtomn and
`C-terminal peptide
`
`CGRP
`
`J - non
`coding
`
`5 '- non
`coding
`
`S i g nd
`sequence
`
`Exon
`
`1
`
`j
`
`ι •
`
`Exon
`
`1
`
`2
`
`Calcitonin
`
`C G R P -I
`
`( a)
`
`C G R P-II
`
`(β)
`
`12 3
`
`A
`
`1 2 3 5
`
`6
`
`12
`
`3
`
`5
`
`Calcitonin/
`CGRP
`gene - I
`
`Calcitonin/
`CGRP
`g e ne - II
`
`Mature
`m R NA
`
`Η
`
`500 nucleotides
`
`FIGURE 1. General organization of the calcitonin/CGRP-I
`in humans. The
`and -II genes and mature messenger RNA transcripts
`exons are indicated in black, the presumed pseudogene corresponding
`to exon 4 in the calcitonin/CGRP-II
`gene as a hatched bar, and the
`mature mRNAs as open bars. From J. Fisher et al.3
`
`ble of activating KA TP channels of vascular smooth
`m u s c l e .1 9 , 3 1'4 2 - 45 T he effects of C G RP on bone are proba­
`bly exerted via the calcitonin receptors. The high affinity
`C G RP specific binding sites, with different levels of
`cross-reactivity with calcitonin or salmon calcitonin,
`have been found in central nervous system, pancreas,
`lung, bone, breast, and lung cancer cells. Specific C G RP
`receptors identified in coronary and other visceral ves­
`sels and heart are linked to c A MP but do not show
`cross-reactivity with c a l c i t o n i n s .1 9'2 1 , 2 4'4 4'45 The regula­
`tion of C G RP secretion is different from that of calci­
`tonin. Intravenous calcium injections in normal h u m an
`subjects as well as in normal rats, cause a six- to seven­
`fold stimulation of calcitonin and carboxyl
`terminal
`flanking peptide (PDN-2) to be cleaved from a calci­
`tonin precursor, with a concomitant marginal or zero
`increase in the plasma C G RP l e v e l s .3 1 , 4 6'47 However, in
`some patients with M T C, infusion of calcium was a po­
`tent stimulus to C G RP release, increasing plasma levels
`two- to
`five-fold. Also, an increase in the cytosolic cal­
`cium concentration is able to stimulate the release of
`C G RP from a h u m an thyroid medullary carcinoma cell
`line, which, on the other hand, does not respond to
`increases in the extracellular calcium concentration.31
`The origin of circulating C G RP remains unclear, ex­
`
`cept for medullary thyroid carcinoma, which is charac­
`terized by high C G RP concentrations
`in tissue and
`plasma. A relatively short plasma half life of about 10
`min and lack of specific organ arteriovenous differences
`indicates a widespread release and degradation of
`C G RP in m a ny tissues. S o me authors postulate that in
`most circumstances the blood levels of C G RP represent
`a spillover phenomenon related to the release of the
`peptide from nerve terminals to promote vasodilatation
`or other f u n c t i o n s .2 4 , 3 1'3 3'4 8'49 The dense distribution of
`C G RP rich structures and the expression of m R NA in
`the central nervous system suggest that C G RP has a
`neuromodulator or neurotransmitter role not limited to
`vasoregulatory effects only, but like calcitonin, extends
`its action to physiologic, metabolic, and behavioral func­
`t i o n s .5 0'51 These and other functions, including sensory
`transmission and motor and autonomic control in many
`organs, are important implications of C G RP regulatory
`effects, but are beyond the scope of this review.
`
`P I T F A L LS OF C G RP M E A S U R E M E N TS IN
`B L O OD A ND TISSUES
`
`The immunochemical heterogeneity of the C G RP mole­
`cule and the specificity of the antisera used for radioim­
`munoassay may account for discrepancies in the re-
`
`hCGRP
`
`rCGRP
`
`I
`
`II
`
`I
`
`II
`
`s —,
`,— s —
`i
`I
`Ala-C^s-Aap-Thr-AJa-Thr-C^s-VaJ-Thr^
`7 θ 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
`1
`2
`3
`4
`5
`6
`- A sn
`Met
`-
`Ser
`
`Ser
`
`- A sn
`
`Asp
`
`-
`
`-
`
`- Qlu
`
`-
`
`-
`
`Ser
`- A sn
`Asp
`-
`FIGURE 2. Amino acid sequences of human and rat CGRP-I and -II or a and β. From H. Morris et al.7
`
`3
`
`

`

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`
`tissue or circulating C G RP under
`levels of
`ported
`experimental conditions, in healthy subjects as well as in
`patients with clinical abnormalities. The metabolic de­
`gradation of C G RP or its precursors, as well as the dif­
`ferent origins of secreted C G R P, may generate various
`molecular species not uniformly detectable by most of
`the specific antibodies. The heterogenous nature of cir­
`culating CGRP-like immunoreactivity (CGRP-LI) was
`shown
`in several experiments, including studies on
`h u m a n s .5 2 - 55 For example, stimulation of the gastroin­
`testinal tract ( 1 4 00 k c a l / m e a l) in healthy subjects, and
`of thyroid C-cells in patients with M T C, resulted in the
`release into the circulation of various molecular weight
`forms of C G R P .52 T he monomer C G R P( 1_3 7) accounted
`for only a fraction of the total immunoreactive C G RP
`measured in blood, and to demonstrate this diversity of
`circulating forms of C G R P, two different antibodies
`were used in the above study, one against the C-ter-
`minal fragment of rat C G R P( 2 8_37 ), and the other recog­
`nizing the whole intact molecule of h u m an C G R P( 1_3 7 ).
`The recently developed immunoassay involving double
`antibodies recognizing different parts of the molecule,
`offers more sensitive and specific alternatives to the
`measurement of circulating C G RP than using previ­
`ously reported r a d i o i m m u n o a s s a y s .7'4 5 , 5 5 - 59 The distri­
`bution of C G RP in different organs has been measured
`in rats and shows the highest concentration in the thy­
`roid and pituitary glands in the range of hundreds of
`picomoles/gram, that is, 10- to 100-fold higher than in
`other endocrine t i s s u e s .4 5 , 6 0 , 61 The thyroid C G RP is co-
`localized with calcitonin in the thyroid C-cells but at less
`than 5% of calcitonin l e v e l s .1 2 , 6 2 , 63 The rat plasma levels
`of C G RP did not show a sex difference or significant
`correlation with concentration
`in different
`tissues.
`There is little information on the distribution of C G RP in
`normal h u m an tissue or organs, and it is limited mostly
`to concentration in malignant tumors.45
`
`in N o r m al S u b j e c ts T he first assessment of
`C G RP
`C G RP circulating in the blood of healthy h u m an sub­
`jects was published by Girgis et al in 1 9 8 5 .51 In unex-
`tracted plasma, the mean levels of CGRP-like i m m u n o­
`reactivity
`(CGRP-LI) measured with specific rabbit
`antibody was 9 4 . 7p ± 6 p g / m L, and was not different
`in men and women. The levels in unextracted plasma
`reported by other authors fell in a close range, and the
`average values in different groups of normal subjects
`l).25,48,64-67
`were between 81 and 140 p g / mL (Table
`Unextracted serum tends to show somewhat higher
`levels, and ranges between 137 to 3 67 p g / m L .6 8 - 70
`Interestingly, our measurements of plasma and serum
`C G R P - LI
`in those same subjects showed
`three-fold
`higher serum concentrations, confirmed in two assays
`using different antibodies (Figure 3 and Table 1). Using
`different extraction procedures it was demonstrated that
`most of plasma or serum immunoreactive material is not
`
`coeluted with synthetic or radiolabeled C G R P, and the
`recovery of endogenous peptide is in the range of about
`1 0 %. In concordance with that finding, most of the re­
`ported C G RP levels in extracted plasma or serum s h ow
`significantly lower concentrations than w h en measured
`in direct assay using unextracted samples, and the aver­
`age values reported for different groups of healthy sub­
`jects were between 2 and 44 p g / mL (Table i ) . 5 6 , 6 2 , 7 i - 77
`However, occasionally C G RP levels in extracted serum
`of normal healthy subjects were found to be m u ch
`higher, up to 3 57 p g / m L .4 6 , 7 6 , 79
`Most authors report no difference in C G R P - LI levels
`between m en and w o m e n. However, there are reports
`showing significantly lower plasma values in m en than
`in age-matched w o m en w h en samples were drawn ran­
`domly during the menstrual c y c l e .4 6 , 67 In addition, in
`w o m en taking the contraceptive pill C G R P - LI
`levels
`were 5 0% higher than in the w o m en not taking the pill
`(P <
`.01) or in m en (Ρ <
`. 0 0 1 ) .67 T he significance of this
`data is difficult to interpret since a possible h o r m o n e-
`dependent volume expansion could stimulate a C G RP
`rise in w o m en using a contraceptive regime, but it does
`not explain the higher values in healthy w o m en υ men.
`
`B l o od a nd T i s s ue C G RP in T u m o rs T he medullary
`thyroid carcinoma cells are a source of large amounts of
`calcitonin and its gene-related peptide, C G R P, in both
`h u m a ns and r at s .3 5 , 4 7 , 5 6 , 6 2 , 8 0 - 82 Calcitonin level in M TC is
`so uniformly and markedly elevated that it became a
`routine test to confirm the d i a g n o s i s .1 7 , 4 7 , 56 C G RP blood
`levels, on the other hand, are 10- to 100-fold lower than
`calcitonin levels, which probably contributes in part to
`the inability of some of the currently used methods to
`demonstrate increased C G R P - LI in patients with M T C,
`leading to conflicting reports. An example of these meth­
`odological difficulties is the report demonstrating that in
`20 of 21 patients with M T C, C G R P - LI levels in direct
`assay were below 3 00 p g / m L, similar to the control
`group of normal subjects. Only one patient had a
`C G R P - LI level as high as 9 42 p g / m L .66 On the other
`hand,
`it was shown by other authors that plasma
`C G R P - LI level were markedly elevated ranging from
`128 to 2 0 10 p g / mL (normal level 12.7 p g / m L) in all five
`preoperative patients with M TC and in 10 of 17 postop­
`erative patients with possible recurrence.75 A provoca­
`tion test with calcium and pentagastrin, a stimulator of
`C-cells in the thyroid gland, showed an increase in
`C G R P - LI l e v e l s .2 7 , 8 3 - 85 However, as expected, the ele­
`vation rate of C G R P - LI was lower than for calcitonin,
`but was particularly high in patients with well-differen­
`tiated M TC and ranged from 2.8 to 23.3 p g / m L. In
`another study, C G R P - LI values in untreated patients
`with M TC were in the 18 to 5 19 p g / mL range, median
`26.9 (normal range, 1.5 to 17, median 3.0), and in
`treated patients with gross persistent or
`recurrent
`tumors were 18 to 647.9 p g / m L, median 8 7 . 9 .56 Injec-
`
`4
`
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`
`TABLE 1. CGRP-LIKE IMMUNOREACTIVITY IN HUMAN PLASMA AND SERUM
`Control Subjects
`
`Patients
`
`Authors, Year
`
`Source
`
`Ν
`
`pg/mL
`
`Ν
`
`pg/mL
`
`Diagnosis, Comments
`
`Normal Subjects
`Girgis et al,
`198551
`Schifter et al,
`198970
`Trasforini et al,
`199276
`Preibisz et al,
`1992§
`
`Valdermarsson
`et al, 199067
`
`Malignant Tumors
`Mason et al,
`198662
`Kim et al,
`1 9 8 766
`Takami et al,
`199075
`Carter et al,
`199156
`Shulkes et al,
`1 9 9 1"
`Schifter et al,#
`198968
`CHF and
`Myocardial
`Infarction
`Edvinsson et al,
`1 9 9 0i 34
`Ferrari et al,#
`199171
`
`Mair et al, 199074
`Hypertension
`Hvarfner et al,
`198846
`Resnick et al,
`19891 38
`
`Jian et al, 198972
`Edvinsson et al,
`198964
`Schifter et al,
`199169
`
`plasma
`
`serum
`
`
`
`plasma* plasma*
`
`serum
`plasma
`serum*
`plasma*
`serum
`plasma
`
`plasma plasma
`
`plasma*
`
`plasma
`
`plasma*
`
`plasma*
`
`plasma*
`
`serum
`
`plasma
`
`plasma*
`
`plasma*
`
`serum*
`
`serum
`
`51
`
`232
`
`8
`
`24
`24
`14
`14
`10
`10
`14
`13
`10
`
`23
`
`17
`
`51
`
`31
`
`?
`
`22
`
`31
`
`16
`18
`
`12
`
`94.7 ± 4.5
`137.5 ± 23
`
`44.3 ± 2
`68.6 ± 6
`367 ± 39
`127 ± 22
`19.8 ± 3
`14 ± 2
`259 ± 38
`81 ± 12
`97.3 ± 13
`192.9 ± 25
`289.9 ± 23
`
`< 3 7 . 9J
`
`132 ± 77
`
`12.7 ±3
`
`3.0
`
`< 76
`
`136.4 ± 10
`
`132.6 ± 15.2
`
`8
`10
`
`5.8 ± 1.7
`
`34
`
`326 ± 44
`
`plasma*
`plasma
`
`serum
`
`18
`31
`
`33
`
`32.9 ± 1 1 .7
`132.6 ± 15.2
`
`137.2 ± 3 3 .7
`
`Noon
`Midnightf
`Aby: 1-61 (Cornell Univ.)
`Aby: 1-61 (Cornell Univ.)
`Aby: 1-61 (Cornell Univ.)
`Aby: 1-61 (Cornell Univ.)
`Aby: RK-6000 (Peninsula Labs)
`Aby: RK-6000 (Peninsula Labs)
`Mentf
`Women (v men)
`Women taking
`contraceptivesjj-(-
`
`19
`
`20
`1
`22
`
`33
`
`1
`
`22
`
`12
`15
`30
`
`18
`
`42
`18
`16
`10
`18
`17
`16
`35
`
`431.9
`
`Medullary thyroid carcinoma
`
`142 ± 193
`942
`128-2010
`
`Medullary thyroid carcinoma
`
`Medullary thyroid carcinoma
`
`6.1-87.9
`
`Medullary thyroid carcinoma
`
`3929
`
`Prostate adeno-carcinoma
`
`177.7 ± 21
`
`Lung carcinomas
`(non-small-cell)
`
`127.7 ± 19
`172.8 ± 17
`
`31
`21
`10.4
`
`CHF, NYHA class II
`CHF, NYHA class, II-IV
`Indian controls
`Italian controls
`Untreated C H F u!
`Treated C H F u!
`Myocardial infarction!!
`
`357 ± 54
`
`Normals and borderline EHT
`
`280 ± 38
`471 ± 67
`89 ± 29
`563 ± 124
`2.3 ± 0.3
`83.4 ± 11.0
`60.6 ± 8
`141 ± 27
`
`EHT
`EHT, high salt diet!!!
`EHT, low salt d i e t ! !!
`Primary aldosteronism!!
`EHT
`HTN untreated!
`HTN after treatment!!
`EHT
`
`5
`
`

`

`TABLE 1. CGRP-LIKE IMMUNOREACTIVITY IN HUMAN PLASMA AND SERUM
`Patients
`Control Subjects
`
`(continued)
`
`Downloaded from https://academic.oup.com/ajh/article-abstract/6/5_Pt_1/434/137281 by University of Wisconsin-Madison Libraries user on 21 March 2019
`
`pg/mL
`
`Diagnosis, Comments
`
`185.8
`227.3
`225.2
`242.5
`121.2
`
`227
`341
`455
`349
`182
`
`174.3 ± 7.2
`223.6 ± 11
`276.6 ± 23
`291 ± 257
`364 ± 166
`
`266.4 ± 62.5
`323.2 ± 66.7
`433.8 ± 9 5 .9
`197 ± 42
`542 ± 53
`
`58.4 ± 19
`45.8 ± 16
`14.9 ±3
`
`32.3 ± 2
`137.5 ± 10
`
`Pregnancy week 10 - 12 jj
`2 0 - 2 2 j jj
`3 0 - 3 2 | tt
`3 6 - t e r m j tt
`
`Postpartum
`Healthy women BC
`Pregnancy month 3 j j
`month 6 jj
`month 9 jj
`Postpartum 24 h jj
`5 days
`Average 24 h level
`3 h post-furosemidejjj
`0.9% NaCl Infusion
`Minor disorders
`Moderate hepatic cirrhosis jj
`Severe hepatic cirrhosis jj
`Hepatic cirrhosis, ascites
`Hepatic cirrhosis, hepatorenal
`syndromejj
`Hemodialysis, start
`ultrafiltrationj
`dialysis
`Fluid overload < 6% j
`7 - 1 3 % t tt
`
`PS - general anesthesia
`PS - epidural analgesia
`Sepsisjjj
`
`Obesityjjj
`High fat meal j
`
`Odar-Cederloef
`et al, 199178
`Miscellaneous
`Bythell et al,
`i 48
`Joyce et al,
`199073
`Zelissen et al,
`199177
`Aby, antibody; EHT, essential hypertension; HTN, hypertension of different etiology; CHF, congestive heart failure; NYHA, New York Heart
`Association; PS, pelvic surgery; BC, before conception.
`* Extracted plasma or serum.
`X Undetectable levels.
`# Serum level calculated from the published figure.
`§ Unpublished data.
`j Ρ < .05, compared to normal subjects or to baseline.
`jj Ρ < .01, compared to normal subjects.
`j jj Ρ < .001, compared to normal subjects.
`
`tion of pentagastrin in M TC patients, as in the previous
`report, increased the C G R P - LI levels, but less than it did
`calcitonin values. In patients w ho were operated on and
`still had elevated calcitonin levels but no detectable
`tumors, C G R P - LI values were lower than in the preoper­
`ative group, < 1 .5 to 23.9 p g / m L, median 6.1. In one
`report, C G R P - LI in the plasma of normal subjects was
`under the assay detection limit (37.9 p g / m L) but, in
`contrast, 19 of 21 patients with M TC had elevated
`
`C G R P - LI levels ranging from 53 p g / mL to 18,437 p g/
`mL, which significantly correlated with increases in cal­
`citonin.62 Calcium or pentagastrin
`infusion also
`in­
`creased C G R P - LI levels two- to five-fold in M TC and
`patients with the highest C G R P - LI levels had particu­
`larly aggressive tumors. However, w h en plasma from
`two patients was studied using H P L C, it showed the
`presence of two molecular forms, neither of which coe-
`luted with synthetic h u m an C G R P. The original human
`
`Authors, Year
`
`Source
`
`Ν
`
`pg/mL
`
`Pregnancy and
`Volume Changes
`Stevenson et al,
`198625
`
`plasma
`
`12
`
`85.2
`
`Saggese et al,#
`199079
`
`serum*
`
`26
`
`170
`
`De Los Santos
`et al, 19911 45
`
`Bendtsen et al,
`199148
`
`Gupta et al,
`199265
`
`plasma
`
`
`
`plasma plasma
`
`10
`10
`10
`232
`
`113.7 ± 61
`223.6 ± 34
`151.6 ± 19
`140.2 ±2
`
`
`
`plasma plasma
`
`10
`
`143 ± 54
`
`Odar-Cederloef
`et al, 19891 44
`
`plasma*
`
`Ν
`
`12
`10
`11
`13
`9
`
`8
`29
`6
`7
`9
`
`12
`
`1
`
`9
`
`8
`
`9
`
`
`
`plasma* plasma*
`
`?
`
`121.2 ± 35
`
`plasma
`
`plasma*
`
`plasma*
`
`26
`
`112
`
`2.0 ±
`
`.3
`
`21.8 ±8
`
`15
`5
`
`7
`7
`16
`
`24
`
`6
`
`

`

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`
`500
`
`400
`
`300
`
`Ε 200
`
`100
`
`495 IIP
`
`164 ipp 11
`
`immunoreactivity
`FIGURE 3. Plasma and serum CGRP-like
`measured by direct radioimmunoassay
`in 10 healthy
`subjects.
`Open bars, plasma; hatched bars, serum. From author's unpub­
`lished data.
`
`C G R P( 1_3 7) sequence was synthesized from thyroid C-
`cells and one can expect at least partial chromatographic
`identity of C G R P - LI extracted from M TC patients. But
`considering the wide range of circulating molecular
`forms of C G R P - LI that require more than one antibody
`to be fully recognized in plasma or serum, the above lack
`of ability to demonstrate the identity of extracted mate­
`rial with synthetic C G RP is not surprising. The review of
`circulatory calcitonin and C G R P - LI data indicates that
`as the calcitonin assay is the preferable test for M T C,
`C G RP may be also a humoral marker of M T C, and mea­
`surements of C G R P - LI have a possible role in predicting
`metastasis and in assessment of m a l i g n a n c y .6 , 1 4'1 7 , 8 6 - 90 It
`is interesting that in patients with medullary thyroid
`carcinoma or other tumors, very high C G RP levels do
`not produce symptomatic hypotension; only some pa­
`tients present with episodes of flushing or diarrhea.6 2'91
`The possible explanation for this fact would be the het­
`erogeneity of C G RP molecular structures,
`including
`both inactive forms and those interacting only with spe­
`cific selected t i s s u e .1 0 2 , 1 03 Another hypothesis is the grad­
`ual down-regulation of C G RP receptor as the peptide
`level slowly increases with growth and spread of the
`t u m o r .1 7'74
`Several studies also demonstrated unequivocally the
`expression of C G RP in other than h u m an thyroid tumor
`cells, that is, in bronchogenic lung cancer, promyelo­
`c y te leukemia, insulinoma, carcinoid, pheochromocy­
`toma, prostatic adenocarcinoma, parathyroid adenoma,
`and in a variety of neoplasmatic cell l i n e s .3 8 , 6 3'7 5'8 6'9 2 - 1 02
`However, in these disease states, due to either low ex­
`pression of C G RP gene or limitations in the immuno­
`chemical method sensitivity, C G R P - LI is not detected as
`uniformly as in M T C. Investigation of 22 patients with
`non - small-cell lung carcinoma showed that pre- and
`post-operative levels of serum C G R P - LI were signifi­
`cantly elevated compared to age-matched normal sub­
`j e c t s .1 33 However, in tumor tissue from the same pa­
`tients no expression of the C G RP gene could be found,
`
`as opposed to cell lines cultured from lung carcinomas.
`T he authors relate the elevated circulating levels of
`CGRP-LI, in the absence of tumor synthesis, to the in­
`creased C G RP release from the vascular sites as the
`response to the altered cardiovascular hemodynamics
`and pulmonary function. In one case of prostatic adeno­
`carcinoma the increased expression of C G RP gene was
`demonstrated in the tumor itself and in liver metasta­
`s i s .3 5'93 Plasma C G RP levels measured repetitively over
`the 3 month period showed the progressive increase,
`parallel with clinical deterioration, from 269 to 3929
`p g / mL (normal < 76 p g / m L ). High concentrations of
`C G R P - LI (> 80 n g /g wet tissue) were also detected in
`five cases of pheochromocytoma, but circulatory levels
`of C G RP were not measured.63
`Based on the limited data available on the plasma or
`serum C G RP levels and CGRP-like immunoreactivity in
`normal and altered homeostasis, it seems premature to
`conclude to what extent these measurements are helpful
`in the interpretation of the C G RP role in various patho­
`logic conditions. Nevertheless, some interesting trends
`in the behavior of circulating C G RP merit attention and
`will be presented below. Table 1 summarizes also most
`of the published data on C G RP blood levels.
`
`C G RP A ND C A R D I O V A S C U L AR S Y S T EM
`
`Calcitonin gene-related peptide is the most potent vaso­
`dilating substance known
`to us, and
`femtomolar
`amounts of this peptide injected in animals or humans
`induces profound vascular r e s p o n s e .6'8'9'1 0 4 , 1 0 6'1 41 Immu­
`nochemical studies have shown that C G RP is distrib­
`uted broadly within the cardiovascular system as a
`dense peripheral sensory network that innervates the
`arteries, veins, heart, and v i s c e r a .3 3 , 5 8 , 1 0 7 - 1 10 Receptors
`for C G RP have been identified both in media and intima
`of resistance vessels.44 The C G RP
`immunoreactive
`fibers penetrate the heart muscle itself, including the
`nodal and conductive system, and the pacemaker cells.
`Atrial myocardium has the highest density of CGRP
`fibers as visualized by immunohistochemistry and by
`determinations of
`the content by
`radioimmunoas­
`s a y .1 1 1 1 12 Distribution of the CGRP-rich fibers in heart,
`blood vessels, and nerve terminals parallels in most
`structures the distribution pattern of substance P, an­
`other neuropeptide with vasodilating activity. How­
`ever, in contrast to substance P, the kidney vessels con­
`tain exclusively C G R P, and the dense rich innervation
`of large veins shows distal reduction in C G RP immuno­
`r e a c t i v i t y .1 0 9 , 1 13 In the experimental animals, infusion of
`C G R P, besides causing a decrease in systemic vascular
`resistance,
`increased the rate and force of contrac­
`t i o n .2 0 , 2 2 , 7 3 , 1 1 4 , 1 15 The hypothesis that there are specific
`receptors in the heart and that there is in vivo inotropic
`C G RP activity still needs further confirmation.
`In healthy h u m an volunteers, systemic infusion pro­
`ducing a 2 0% increase above the physiologic levels of
`
`7
`
`

`

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